biology 3492: - tgd | tetrahymena genome database …ciliate.org/files/chalker protocols/molecular...

Biology 3492 Spring 2008

Biology 3492:Laboratory experiments with eukaryotic microbes:

Functional genomics using Tetrahymena in the classroom

Contents: Pages1. Bioinformatics Module: 2 - 7

-The Tetrahymena genome database (TGD): getting your gene’s sequence-Bioinformatics analysis of Tetrahymena genes

2. GFP-fusion protein localization module: 8 - 27-Overview-Gene Amplification and cloning-Clone verification-Fluorescent protein gene fusion construction-Tetrahymena transformation-Fluorescent protein gene fusion visualization

3. GFP-fusion protein expression module: 28 - 31-Protein isolation-Western blotting and detection

4. Gene expression module: 32 - 39-RNA isolation-Reverse transcriptase PCR

5. Appendices 40 - 67-Appendix 1: Sample syllabus 40-42-Appendix 2: Working with Microbes Intro Lab 43-50-Appendix 3: Assignments, problems sets, and grading sheets 51-67

Biology 3492 Spring 2008Bioinformatics Module

------------------------------------------------------------------------------------------------------------The Tetrahymena genome database (TGD): getting your gene’s sequence------------------------------------------------------------------------------------------------------------This semester we will be working together to study a (specific biological process) in the ciliated protozoan, Tetrahymena thermophila. (Add in sentence or two describing the process and its significance to biological function). As our starting point, we will look for genes within the Tetrahymena genome that are homologous to genes known to encode proteins involved in this process. (Alternatively, one could start with a list of proteins found through proteomic analysis) One way to find such genes is to look at the genome of a well studied organism such as baker’s yeast, Saccharomyces cerevisiae, (http://www.yeastgenome.org/). Use the GO annotations to find know genes in a process of interest, then use those protein sequences in BLASTp searches against the Tetrahymena genome.

You will each be assigned one gene to characterize during the course. We will use the Tetrahymena genome database (www.ciliate.org) to find our genes’ and the proteins sequences they encode, download information to files for use throughout the course, and design oligonucleotide primers to amplify and clone the genes from Tetrahymena genomic DNA. We will also design PCR primers to each gene to perform rtPCR to determine whether and when these genes are expressed. In this first week of class, we will retrieve our gene sequences and design the primers for this work. So each one will design primers for the chosen gene for both cloning and expression analysis. We will use the Primer3 program (http://frodo.wi.mit.edu/), which matches optimal primer pairs for PCR. We will also become familiar with the program Gene Construction Kit, which we will use to analyze and display our genes’ sequences. A graphic representation of a DNA sequence is an important part of the design of experiments in molecular biology. One needs to be familiar with the overall structure/layout/features of the DNA of interest to enable future studies.

Steps: 1. Getting your gene sequence.Go to TGD and download the sequence of your gene of interest.--Download your: 1) coding sequence

2) ORF translation 3) at least a 5 kbp region spanning your gene sequence (larger if this does not include your whole gene.

-- Paste each sequence into a Word file (save your work)-- Paste your genomic sequence (3) into a GCK file

MAKE SURE TO SAVE YOUR WORK

2. Annotating your gene sequence.a. Color your coding region Blueb. Find your introns, Compare your Genomic sequence to your coding sequence using the EMBOSS alignment website (http://www.ebi.ac.uk/emboss/align/)The introns will we the gaps in the coding sequencec. Go to the TGD genome browser and look for available cDNA sequences. Download these and use to confirm the predicted introns. Color predicted introns RED, confirmed introns GREEN.d. Mark the following restriction sites on your sequence: ApaI, BamHI, BglII, BsrGI, EcoRI, EcoRV, HincII, HindIII, NotI, PstI, SacI, SalI, ScaI, XbaI, XhoI

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http://www.ebi.ac.uk/emboss/align/

http://www.ciliate.org/

Biology 3492 Spring 2008Bioinformatics Module3. Designing oligos to clone your gene sequence.

To get started, one needs to first PCR amplify one’s desired coding sequence and clone into an entry vector such as pENTR-D. When using the pENTR-TOPOD cloning kit, 4 nucleotides –CACC- must be added to the primer in frame with the ATG start codon (plus the six nucleotides upstream of the ATG start codon sequence to ensure good translation of the mRNA). This will allow directional cloning into the pENTR-D vector.

your favorite gene- PCR amplify and clone into pENTR-D-TOPO

a. Paste in the your coding sequence into the primer3 dialog box. Set the size of your PCR product to amplify between the size of your coding sequence and this minus 50 bases.

b. Adjust parameters as needed to get appropriate oligos. The upstream oligo must start at –6 plus have a CACC added to the 5’ end. The downstream oligo must start at the last base of the last codon and be designed anti-sense to the gene. A GATATC should be added to its 5’ end.

c. To design primers for expression analysis, if possible locate an intron in your gene and design them to span the intron. Paste this region of your sequence into the Primer3 dialog box. Set size range of your product to be between 170 and 250 bp (not counting the intron). Select primers.

d. Copy all primer sequences into your word file and email to Prof. Chalker ([email protected]).

MAKE SURE TO SAVE YOUR WORKAccessing Bio3492 folder on the NSLC server (to use to turn in computer files):In ‘Finder’ select ‘GO’:’Connect to Server’Select ‘Local’ folder: Select ‘NSLC Server’: Connect’: logon as ‘biol3492’ password: XXXXXXCreate a folder of your files on your computer’s desktop; before ending, be sure to Click and drag your folder into for future use.

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yfg coding sequence

CACC XXX XXX ATG................................................................................................GATATC-6 +1

Biology 3492 Spring 2008Bioinformatics ModuleSome GCK function commands you will need:Make codon table for TetrahymenaOpen file; Construct: features :edit codon table: new codon table,Change TAA and TAG stop codons to Gln (glutamine); name ‘Tetrahymena’ ; ‘OK’

Select codon table for TetrahymenaOpen file; Construct: features :select codon table: Select ‘Tetrahymena’ ; ‘OK’

To show numbered coordinates of the sequenceOpen file Construct: Display: Show Positions

To group sequence for friendly displaySelect sequence; Format:Grouping:(select ‘by threes’ for protein sequence, ‘by tens’ for others)

To color a block of sequenceSelect sequence; Format:color:(select color)

To designate a regionSelect sequence; Construct: features :Make region

type in name, designate as protein if desired

To change regions arrow display to line or different arrowSelect region; Format: lines: (select line type)

To mark restriction sites deselect all sequence; Construct: features :mark sites

in popup window, select enzyme names one at time and ‘add’ to list, click ‘OK’

To mark a particular position (e.g. to indicate an oligo position)Select cursor location at site; Construct: features: mark location

To insert a particular sequence into an existing file (e.g. to indicate an oligo position)Select sequence to be inserted and copyPlace cursor over at site to insert sequence and paste sequence to this location Notes: By highlight a region of sequence in target file, the highlighted sequences will be removed during operationSequence can be inverted by ‘special paste’

To space restriction site markersSelect sites in graphic display Format:Sitemarkers:automatic arrangement (or just drag individually)

To generate list of restriction sites in your sequencedeselect any sequence; Construct: features :list sitesin popup window, select enzyme names one at time (or ‘add all’ and ‘add’ to list, click ‘OK’

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Biology 3492 Spring 2008Bioinformatics Module

Database and analysis tools links

Tetrahymena Genome Databasewww.ciliate.org

Eric Cole’s: biology of Tetrahymena websitehttp://www.stolaf.edu/people/colee/

Sequence analysis tools at EMBL-EBIhttp://www.ebi.ac.uk/Tools/sequence.html

EMBOSS pairwise alignmentshttp://www.ebi.ac.uk/emboss/align/

ClustalW sites for multiple sequence alignmentshttp://www.ebi.ac.uk/clustalw/

http://clustalw.genome.jp/[Easier to download trees]

NCBI Blast homepagehttp://www.ncbi.nlm.nih.gov/BLAST/

Pfam database of conserved protein motifshttp://pfam.wustl.edu/

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http://pfam.wustl.edu/

http://www.ncbi.nlm.nih.gov/BLAST/

http://clustalw.genome.jp/

http://www.ebi.ac.uk/clustalw/

http://www.ebi.ac.uk/emboss/align/

http://www.ebi.ac.uk/Tools/sequence.html

http://www.stolaf.edu/people/colee/


Biology 3492 Spring 2008Bioinformatics Module-----------------------------------------------------------------------------------------------------------------Bioinformatics analysis of Tetrahymena genes-----------------------------------------------------------------------------------------------------------------Understanding what’s in a sequence.Once a gene is identified, one of first steps is trying to understand the biological function of its encoded protein is to use bioinformatics to look for proteins of known function that have shared structure. A number of bioinformatic tools exists to assist scientists in such analyses.

A. One of the first tests one will want to do is see what proteins exist in other organisms that have sequence similarity to your protein. These could be direct homologues or ones with shared functional domains. This is a simple analysis using “BLAST” tools that perform pair-wise comparisons of a query (your proteins sequence) against all the know protein sequences in the genbank database. Go to the BLAST homepage:NCBI BLAST homepagehttp://www.ncbi.nlm.nih.gov/blast/Blast.cgi

Select ‘protein BLAST’, paste in your protein sequence and search. Does the BLAST search identify any conserved protein domains? Note their location in your protein.Look at your ‘hits’. Is there a common class of proteins that are similar to your protein sequence? Save your search as a ‘Page Source’ for future reference

B. Homology suggest that proteins share function, but not necessarily that they are involved in the same process. Orthologs are proteins that appear to be the same protein in different organisms that share a direct line to a common ancestoral protein (and thus are likely doing the same thing). Organisms that share closer evolutionary histories are more likely share orthologous proteins. A database of ciliate orthologs has been develop to assist in this analysis. Ciliate Ortholog Databasehttp://oxytricha.princeton.edu/COD/

Select ‘BLASTO’, paste in your protein sequence and search. Does your protein fall into a ortholog group? Save your search as a ‘Page Source’ for future reference.If you have a direct ortholog group, select the ClustalW alignment display. Save for future reference,

C. While BLAST searches allow us to find homologs as well as conserved protein domains, any one database can miss important information. The SMART database is a useful tool to look for conserved domains, not-so-conserved domains, and other functional motifs in one’s protein sequence. SMART modular domain databasehttp://smart.embl-heidelberg.de/

Paste in your sequence and search for: Outlier homologues, PFAM domains, internal repeats, intrinsic protein disorder. Save you search. If you want to save your diagram, Print and Save as a PDF.

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http://smart.embl-heidelberg.de/

http://oxytricha.princeton.edu/COD/

http://www.ncbi.nlm.nih.gov/blast/Blast.cgi

Biology 3492 Spring 2008Bioinformatics ModuleDatabase links

NCBI Blast homepagehttp://www.ncbi.nlm.nih.gov/blast/Blast.cgi

NCBI Protein BLASThttp://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on

Tetrahymena Genome Databasewww.ciliate.org

Ciliate Ortholog Databasehttp://oxytricha.princeton.edu/COD/

Tetrahymena Genome Blast Searchhttp://tigrblast.tigr.org/er-blast/index.cgi?project=ttg

Paramecium Genome Databasehttp://paramecium.cgm.cnrs-gif.fr/

ClustalW sites for multiple sequence alignmentshttp://www.ebi.ac.uk/clustalw/

http://clustalw.genome.jp/[Easier to download trees]

SMART modular domain databasehttp://smart.embl-heidelberg.de/

Pfam database of conserved protein motifs (two mirrored sites) http://pfam.sanger.ac.uk/ http://pfam.janelia.org/

PubMed (for literature searches)http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed

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http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed

http://pfam.janelia.org/


http://smart.embl-heidelberg.de/

http://clustalw.genome.jp/

http://www.ebi.ac.uk/clustalw/

http://paramecium.cgm.cnrs-gif.fr/

http://tigrblast.tigr.org/er-blast/index.cgi?project=ttg

http://oxytricha.princeton.edu/COD/


http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on



http://www.ncbi.nlm.nih.gov/blast/Blast.cgi

Biology 3492 Spring 2008GFP localization module---------------------------------------------------------------------------------------------------------------Fusion of your favorite gene (yfg) to fluorescent proteins for in vivo localization---------------------------------------------------------------------------------------------------------------To facilitate the fusion to Fluorescent Proteins, we created YFP and CFP expression vectors that contain the Invitrogen Gateway cloning cassette that uses Lambda phage recombinase to transfer DNA fragments from the appropriate TOPO-cloning generated entry vector into the desired Y/CFP destination vector (pICY-gtw or pICC-gtw). (This mitigates the need to do more cumbersome and less efficient restriction enzyme digestion--fragment isolation—ligation mediated cloning). We placed the Gateway cassette upstream of YFP to create this carboxy-terminal YFP-fusion destination vector. Any coding sequence cloned into the appropriate Entry vector such as pENTR-D (TOPO-cloning kit available) can be recombined into this vector and expressed as a YFP fusion under control of the CdCl2 inducible MTT1 promoter.

XhoI PspOMIpICY-gtw MTT1 promoter YFP

Ampicillin resistance gene

To get started, one needs to first PCR amplify one’s desired coding sequence and clone into an entry vector such as pENTR-D. If one uses the pENTR-TOPOD cloning kit, 4 nucleotides –CACC- must be added to the primer in frame with the ATG start codon (plus the six nucleotides immediately upstream the start codon to ensure good translation of the mRNA; alternatively add the sequence AATAAA between the CACC and the ATG ). This will allow directional cloning into the pENTR-D vector. Optionally add GATATC – an EcoRV site) to the 5’ end of the downstream primer. This allows screening for completely amplified PCR products by restriction enzyme digestion prior to DNA sequencing. Once cloned in the pENTR-D vector, this entry vector is mixed with the pICY(or C)-gtw vector plus the LR clonase II enzyme. An room temperature reaction incubated several hours to overnight followed by E.coli transformation into TOP10 cells typically produces >75% correct recombinants.

your favorite gene- PCR amplify and clone into pENTR-D-TOPO

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yfg coding sequence

CACC AATAAA ATG................................................................................................GATATC

Mtt pmr-rRNA

Gateway

3.7 kbp

YFG

Biology 3492 Spring 2008GFP localization modulehttp://tools.invitrogen.com/content/sfs/manuals/pentr_dtopo_man.pdf

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Biology 3492 Spring 2008GFP localization module--------------------------------------------------------------------------------------------PCR amplification Tetrahymena gene fragments--------------------------------------------------------------------------------------------

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GATATC (EcoRV site)

CACCXXXXXX

epiplasm candidate gene

Biology 3492 Spring 2008GFP localization modulePCR works by doing successive rounds of DNA synthesis primed from convergently facing oligonucleotides complementary to a DNA region of interest (See Bioinformatic Module for primer design exercise). Using the oligonucleotide primers complementary to parts of your gene and genomic DNA, we will generate DNA fragments of the coding regions of each. Once we do that, we will clone these into an intermediate plasmid before fusing them to YFP in pICY-gtw.

You will set up three reactions with different amounts of MgCl2 added in ensures getting at least one reaction to work. Also you will want to set up reactions on ice. Always add enzyme (NEB Phusion DNA polymerase) last just before you are ready to put the reactions in the thermocycler. (NOTE: of course, one can use different polymerases if necessary, but a proofreading polymerase is highly recommended).

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Gateway cloning pICY-gtw Spring 2007PCR Reaction conditionsReaction components Amount for 1 rxn ( l)

Rxn1 Rxn2 Rxn3 5x PCR buffer 8.050 mM MgCl2 0.0 1.0 1.510 M upstream oligo 1.010 M downstream oligo 1.010mM dNTPs 0.5NEB Fusion DNA polymerase 0.5 (ADD LAST!)H20 28.0 27.0 26.5Genomic DNA 1.0Total 40 l

Program the thermocycler with the followingPCR cycling conditions:1 cycle 94oC for 4 min.36 cycles 94oC for 30 sec

55-56oC for 30 sec72oC for 1 min to 7 minute (~30 sec-1 min/kbp of fragment)

1 cycle 72oC for 5 min.

Pour a 1% agarose gel to use to visualize reaction products. This will occur next lab period.

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Gateway cloning pICY-gtw Spring 2007---------------------------------------------------------------------------------------------------------Gel electrophoresis of DNA fragments--------------------------------------------------------------------------------------------------------Materials:

agarose solution in TBE (1.5%)1X TBE gel loading dye10 mg/ml ethidium bromide

1. To prepare 50 ml of a 1.0% agarose solution, measure 0.50 g agarose into a glass beaker or flask and add 50 ml 1X TBE.2. Microwave until agarose is dissolved and solution is clear.3. Allow solution to cool to about 550C before pouring. (Ethidium bromide can be added at this point to a concentration of 0.2 g/ml)4. Prepare gel tray by sealing ends with tape or other custom-made dam.5. Place comb in gel tray and pour 50oC gel solution into tray to a depth of about 5 mm. Allow gel to solidify about 20 minutes at room temperature.6. To run, gently remove the comb, place tray in electrophoresis chamber, and cover (just until wells are submerged) with electrophoresis buffer (the same buffer used to prepare the agarose)7. To prepare samples for electrophoresis, add 2 l of 6x gel loading dye and 10 l of the PCR reaction. Mix well. Load entire sample of DNA into well.8. Load 1 kbp DNA marker9. Electrophorese at 100 volts until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized.10. Visualize on UV transilluminator.

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Biology 3492 Spring 2008GFP localization module

---------------------------------------------------------------------------------------------------------------PCR reaction clean-up: removal of primers and dNTPs---------------------------------------------------------------------------------------------------------------PCR reactions contain in addition to the PCR products, salts associated with the PCR buffer, excess dNTPs, and oligonucleotide primers that can interfere with downstream applications. Thus, we want to try to remove these. We will use Montage PCR centrifugal filter devices for this purpose. These are disposable, single-use centrifugal devices for processing aqueous biological solutions in the 0.1 to 0.5 mL volume range. They are used in fixed angle micro-centrifuge rotors that accommodate 1.5 mL microfuge tubes. Used for PCR product purification, Montage PCR devices allow for up to 500 L sample clean-up of salts and primers with a concentration factor of 5X to 10X in 15 minutes, with no solvents or chemicals required. The Montage PCR device consists of a filtrate collection vial with attached cap and a sample reservoir. The sample is spun to “virtual” dryness in a 15-minute spin time, followed by sample reconstitution, and an invert spin transfer into a clean vial for subsequent analysis and/or storage.

Diagram of Montage PCR Centrifugal Filter Device

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BASIC STEPS1. Load PCR sample and 400 µL TE-buffer2. Spin for 15 minutes at 1000 x g


How to Use the Montage PCR Device1. Insert the Montage PCR sample reservoir into one of the two vials provided.

2. Pipette 375 L distilled water into sample reservoir (0.5 mL maximum volume), without touching the membrane with the pipette tip. Add 30L to 50 L of PCR reaction to the reservoir. Seal with attached cap.NOTE: Smaller volumes of PCR product may be used, but the volume in the sample reservoir should always be adjusted to a final volume of 400 L

3. Place assembly in a compatible centrifuge and counterbalance with a similar device.NOTE: When placing the assembled device into the centrifuge rotor, align the cap strap toward the center of the rotor.

4. Spin the Montage PCR unit at 1000 X g for 15 minutes.NOTE: For optimal performance in recovery, do not centrifuge longer than 15 minutes or at greater than 1000 x g as yield loss may occur due to overdrying of the sample.

5. Remove assembly from centrifuge. Separate vial from sample reservoir. Save filtrate until sample has been analyzed.

To recover the purified DNA:6. Place sample reservoir upright into a clean vial and add 20 L distilled water or TE buffer carefully to the purple end of the reservoir. (Avoid touching the membrane surface).

7. Invert the reservoir into a clean vial and spin at 1000 x g for 2 minutes.

8. The Clean PCR product is ready for analysis or for use in the next reaction (Go to pENTR-TOPO/D reaction)

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-------------------------------------------------------------------------------------------------------------------Directional Topo cloning of DNA fragments

------------------------------------------------------------------------------------------------------------------Methods Overview

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----------------------------------------------------------------------------------------------------------------Directional Topo cloning of DNA fragments (continued)

---------------------------------------------------------------------------------------------------------------A rapid way to clone PCR products is using a TOPO cloning kit. One can simply purchase a kit that will take a PCR product and join it to an already prepared plasmid (See description on preceding page). The kit we will use employs the enzyme topoisomerase to join the PCR product to the prepared vector that can then be recovered by transforming the mix into E. coli. We used PfuTurbo DNA polymerase to amplify our PCR products because it makes fewer errors that Taq DNA Polymerase. This TOPO Kit actually clones blunt ended PCR products so we do not need to added additional A’s using Taq DNA polymerase and is designed to clone the products in one orientation only. See previous page.

4.0 l of PCR product1.0 l of salt solution1.0 l of directional Topo vector

incubate reaction 10-20 minutes at room temperature

E. coli transformationWarm selective plates (L + kanamycin) at 37°C for 30 minutes.

• Thaw on ice 1 vial of One Shot® cells for each transformation.

One Shot® Chemical Transformation Protocol1. Add 2 µl of the TOPO® Cloning reaction into a vial of One Shot® Chemically Competent E. coli(Thawed on ice) and mix gently. Do not mix by pipetting up and down.2. Incubate on ice for 5 to 30 minutes.Note: Longer incubations on ice do not seem to have any affect on transformationefficiency. The length of the incubation is at the user’s discretion.3. Heat-shock the cells for 30 seconds at 42°C without shaking.4. Immediately transfer the tubes to ice.5. Add 250 µl of room temperature S.O.C. medium.6. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37°C for1 hour.7. Spread 10-50 µl from each transformation on a prewarmed selective plate andincubate overnight at 37°C. To ensure even spreading of small volumes, add20 µl of S.O.C. medium We recommend that you plate two different volumesto ensure that at least one plate will have well-spaced colonies.8. An efficient TOPO® Cloning reaction should produce several hundredcolonies. Pick ~10 white or light blue colonies for analysis. Do not pick dark blue colonies.

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Screening E. coli transformants for correct plasmid

We will use PCR to determine which E.coli transformants contain clones of our genes. We will do this by lysing the E.coli directly in the tubes and using this as a template for PCR using our gene specific primers we designed (for later use in rtPCR also)

1. Pick E. coli transformant from plate with toothpick

2. Twirl in microfuge tube containing 40ul water to remove cell from toothpick

3. Spot remaining cells from toothpick on master plate grid

4. Cap and heat in PCR tubes to 95C for 5min. (program in thermocycler)

5. Keep lysate on ice until use

6. Use 3 ul of each lysate in PCR reactions for 25 cycles.

PCR Reaction conditionsSet up reactions on ice with everything except for lysate. Make master mix for all reactions, excluding the lysate. Aliquot to PCR tubes and add lysate. Amplify in thermocycler.

Amount for 1 rxn ( l) 10x PCR buffer 3.0 |25mM MgCl2 (if not in buffer) 1.2 |10 M upstream oligo 1.0 |10 M downstream oligo 1.0 |10mM dNTPs 0.3 combine and aliquot 27 l per tubeTaq DNA polymerase 0.25 |H20 20.25 |

Lysate (or genomic DNA control) 3.0Total 30 l

Program the thermocycler with the followingPCR cycling conditions:1 cycle 94oC for 4 min.25 cycles 94oC for 30 sec

56oC for 30 sec72oC for 50 sec

1 cycle 72oC for 5 min.Pour a 1.2% agarose gel to use to visualize reaction products.

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GATATC CACCXXXXXX Candidate geneatt att


------------------------------------------------------------------------------------------------------------------Directional Topo cloning: Plasmid DNA isolation and restriction enzyme analysis

------------------------------------------------------------------------------------------------------------------Eppendorf FASTPLASMID DNA ISOLATION Kit instructions1. Chill the Complete Lysis Solution on ice if necessary.

2. Pellet 1.5 ml of fresh bacterial culture at maximum speed (at least 5,000 x g) for 1 minute in the provided 2 ml Culture Tube.

3. Remove medium by decanting, taking care not to disturb bacterial pellet.

Inverting the tubes on a paper towel may improve removal of the medium.

Note: If necessary, a maximum of 3 ml of culture can be processed.

4. Add 400 ul of ICE-COLD Complete Lysis Solution.Note: The Complete Lysis Solution MUST be ice-cold (0–4°C) to obtain

maximum yield.

5. Mix thoroughly by constant vortexing at the highest setting for a full 30 seconds.This step is critical for obtaining maximum yield.

Note: It is helpful to vortex 15 to 30 second prior to adding lysis solution. If the pellet is not completely resuspended, continue vortexing until the lysate is a homogenous solution with no apparent cell clumps visible. Several tubes may be processed at the same time.

6. Incubate the lysate at room temperature for 3 minutes.Note: The lysate should appear non-viscous and slightly cloudy, with no

precipitate.

7. Transfer the lysate to a Spin Column Assembly by decanting or pipetting.

8. Centrifuge the Spin Column Assembly for 60 seconds at maximum speed.

Note: It is not necessary to decant filtrate after Step 8. Wash Buffer may be addeddirectly to the Spin Column Assembly in the centrifuge.

9. Add 400 ul of DILUTED Wash Buffer to the Spin Column Assembly.

10. Centrifuge the Spin Column Assembly for 60 seconds at maximum speed.

11. Remove the Spin Column from the centrifuge and decant the

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filtrate from theWaste Tube. Place the Spin Column back into the Waste Tube and return it to thecentrifuge.

12. Centrifuge at maximum speed for 1 minute to dry the Spin Column.

13. Transfer the Spin Column to a Collection Tube.

14. Add 50 ul of Elution Buffer directly to the center of the Spin Column membraneand cap the Collection Tube over the Spin Column. WAIT 1 minute.

Note: To avoid inconsistent elution volumes, ensure that the elution buffer is pipetteddirectly onto the surface of the filter, avoiding contact with the wall of the column.

15. Centrifuge at maximum speed for 60 seconds.

16. Remove and discard the Spin Column.

17. The eluted DNA can be used immediately for downstream applications or stored at -20°C.

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-------------------------------------------------------------------------------------------------------------------Directional Topo cloning: Plasmid DNA isolation and restriction enzyme analysis

-------------------------------------------------------------------------------------------------------------------Once we recover plasmid DNA from our, we will digest aliquots with restriction enzymes and analyze the fragment sizes by gel electrophoresis (see Appendix at end of handout and your NEB catalogue for background information on restriction enzymes). Each plasmid will be digested separately with BsrGI and EcoRV separately and possibly one enzyme specific for your gene.

Materials:Plasmid DNA prep10x enzyme buffer (refer to NEB catalog for buffer needed)100x BSAEnzymes (BsrGI and EcoRV and ?)Sterile H2O

Reactions conditions:8 ul of DNA___ ul 10x enzyme buffer ___ ul 100x BSA___ ul Restriction enzyme (5-10 units)___ ul H2O (sterile)20 ul Total VolumeDigest at 37 oC, 45 minutes to 2 hours

Electrophorese samples on 1.2% agarose gel. Confirm sizes based on size of 1 kbp and 100 bp ladders loaded with sample.

If we find the correct clones proceed to LR recombination reactions.

FastPlasmid Mini

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---------------------------------------------------------------------------------------------------------------The L-R recombination reaction---------------------------------------------------------------------------------------------------------------The LR Clonase enzyme is a purified form of phage Lambda recombinase. It will remove the coding sequence from the pENTRTOPOD vector and replace the gateway cassette placed in the new pIGF-GTW vector.

The LR ReactionLR Clonase™ II enzyme mix is supplied as a 5X solution.

1. Add the following components to a 1.5 ml microcentrifuge tube at room temperature and mix: a. 1-3 µl of coding sequence Entry clone (50-150 ng total) b. 1 µl of pICY-GTW or pICC-GTW destination vector (400 ng/µl) c H2O to 4 µl

2. To each sample (Step 1, above), add 1 µl of LR Clonase™II enzyme mix to the reaction Mix well by vortexing briefly twice. Microcentrifuge briefly.3. Return LR Clonase™ II enzyme mix to -20°C storage4.. Incubate reactions at 25°C for at least 1 hour (overnight incubation results in very efficient recovery of recombinants).5. Add 0.5 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes.6. Use 1.5 to 2 ul in electroporation of TOP10 E. coli cells (next page)

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Transformation of E. coli with ligation mix by electroporationFor these recombination reactions, we will use TOP10 electrocompetent cells and electroporation. Electroporation is a very efficient method. This involves mixing DNA with washed E. coli cells and hitting them with an electric charge that causes the DNA in the surrounding medium to enter the cells. Here are the steps:

1. Place electroporation cuvette on ice and thaw an aliquot of electrocompetent cells on ice. This will take ~5 min.

2. Turn the power on for the electroporator. Set the electroporator:2.45 kVolts125 ohms resistance 50 uF capacitance.

3. Put 1 ml of SOC in a sterile tube and draw it up into a sterile Pasteur pipette.

4. Add 1.5 ul of the ligation mix to the thawed cells. Mix by gentle stirring with the pipette tip. (The cells are fragile, do not vortex).

5. Pipette the cells into a clean, cold cuvette and place into the electroporator (there is a notch on the cuvette so that it only fits in one orientation).

6. Push the pulse button! If the cuvette was dirty or there was some salt in the sample, you may get arcing. If everything is OK, the pulse length will go to ~ 6.5 msec.

7. Immediately squirt the 1 ml of SOC into the cuvette and remove the cells to the sterile tube.

8. Incubate the cells, shaking, at 37oC for 1 hour.

9. Spread 200 ul of the transformed cells on LB plates containing the appropriate drug (Ampicillin) for selection.

10. Incubate plates overnight at 37 0C

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-------------------------------------------------------------------------------------------------------------Electroporation transformation of Tetrahymena with YFP-fusion constructs -----------------------------------------------------------------------------------------------------------------

The pICY-gtw plasmid contains the necessary DNA sequences both to replicate and be selected in E. coli and Tetrahymena. This rDNA-based vector can replicate in Tetrahymena as a linear mini-chromosome if it is introduced into Tetrahymena during conjugation, just prior to the time that DNA rearrangement in the developing macronucleus occurs. The circular plasmid is then cleaved at breakage sequences flanking the E. coli vector sequences and is converted to a palindromic linear chromosome. We can select transformants based on the virtue that the 17S rRNA allele in the plasmid confers resistance to the drug paromomycin on the cells. Drug selection of the transformants will need to be done the day after we perform the electroporation.

1. Preparation of cellsWe have to transform Tetrahymena during a narrow window of development, 8.5 to 9.5 hours after initiating conjugation. To prepare the cells for this, the follow has been performed outside of class time.

Day 1 Inoculate 100 mls with 2 mls of CU428 of 1 x SPP or Neff’s medium (+1x penicillin/strepomycin and fungizome), incubate overnight at 30 oC in 1 liter flasks. Repeat with CU427.

Day 2Pour 50 mls of grown culture into each of two 50 ml conical tubes to harvest cells. Centrifuge in swinging bucket rotor @ 2500 rpm for 3 min.Wash cells with 40 mls of 10 mM Tris-HCl (pH 7.5)Harvest cells again by centrifuging in swinging bucket rotor @ 2500 rpm for 3 min.resuspend each tube of cells in 40 mls of 10 mM Tris-HCL. Pour into 150 mm petri dish.Allow to starve at least 8 hours at 30 oC.

Approximately 9 hours prior to electroporation, mix equal numbers of pre-starved Tetrahymena of complementary mating types. Allow cells to pair for conjugation @ 30oC

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P GFP

pIGF-1

P GFP


Day 3 – Electroporation dayMaterials needed:Petri Dishes with Mating Cells (Mixed 9 hours prior)50 ml conical tubes 10 mM Hepes (pH 7.4)Electroporator and Cuvettes1xSPP medium w/ 1x fungizome and 1x penicillin/streptomycin15 ml conical tubes with 5 mls each of medium96 well tissue culture platesMultichannel pipettorMedia ResevoirsPlastic boxes to incubate plates

Procedure:1. Mix 10 to 15 ug of each plasmid DNA to electroporate w/ 10 mM Hepes for at total volume of 50 ul

2. Transfer 40 to 50 mls conjugating cells to 50 ml conical centrifuge tubes (using 25-ml. pipettes) and spin down for 3 minutes at 2500 RPM. Decant supernatant quicklyNote: It is important to decant medium as soon as centrifuge stops as the Tetrahymena will try to rapidly swim out of the pellet.

3. After pouring off supernatant and gently resuspending, add 40 mls of 10 mM Hepes incubate at room Temperature for 5 minutes

4. Harvest the 40 mls of cells again by centrifuging @ 2500 rpm for 3 min.resuspend cells in 400 ul Hepes buffer and proceed with electroporation

5. BTX cell porator: Turn on and set up with capacitance=275µF , resistance=25Ω, Voltage = 250V. Check all settings.

Procedure: For EACH sample of mating cells to be electroporated: 1. Add 200 µl of the mating cells to the DNA in the microfuge tube.2. Mix gently by pipetting up and down.3. Gently Pipette the DNA+cell mixture from the microfuge tube into the cuvette.4. Put the cuvette in the electroporation chamber and push the pulse button. Note time.(4b. Record peak voltage and duration (msec.))5. Remove cuvette from chamber. Press reset button.6. Wait one minute after electroporation, Wash cell out of cuvette with growth medium by

pipetting cells with a a pasteur pipette into a tube containing 5 ml of 1XSpp + 1X pen/strep + 1X fungizone. Alternative, cells can be resuspended directly into a media resevoir and plated.

Repeat the above for all samples. Flush cuvette with water and 70% ETOH to sterilized.

25


AT BENCH AFTER ELECTROPORATION

Plating of Cells:Make 30 mls of a 1:10 dilution of Electroporated cells by mixing 27 mls Spp (+ 1x p/s, 1x fungizome.) and 3 mls of electroporated cellsPour cells into media trayPlate each sample into 3 separate 96 well plates, aliquoting 100 l/well using multichannel pipettors.Incubate at 30 degrees Celsius at least 16 hours before adding drug to select transformants.

On Friday, we will add selection drug to your transformation plates

ON FRIDAY------------------------------------------------------------------------Selecting Tetrahymena electroporation transformants------------------------------------------------------------------------Materials:96 well tissue culture plates containing putative transformants 1xSPP medium200x fungizome stock1000x penicillin/streptomycin stock10 mg/ml paromomycin (Pm) stock 500 ml flasks

Prepare:30 mls of 1x Spp (+1x penicillin/strepomycin and 1x fungizome + 200 g/ml Pm

30 mls 1x Spp___mls Pen/Strep stock___mls fungizome stock___ mls Pm stock

We will have this prepared, but you should calculate out the amounts of drugs neededPour Spp into media reservoiraliquot 100 l/well to each 96 well plate containing cells.Incubate at 30 degrees Celsius at least 3 days before checking for transformants.

26


------------------------------------------------------------------------Maintaining electroporation transformants ------------------------------------------------------------------------Overview of steps

1. Score the number of total transformants2. Select 12 transformants from each shot for future experimentation

For each of our constructs that we have electroporated into Tetrahymena, we want to identify wells containing paromomycin resistant transformants. We will then select a reasonable number (six to twelve, if we generate at least this many) to propagate for further experiments.

Materials:96 well tissue culture plates containing transformants Tissue Culture Plates (24 well)1xSPP medium200x fungizome stock1000x penicillin/streptomycin stock10 mg/ml paromomycin (Pm) stock 500 ml flasks

Prepare:40 mls of 1x Spp (+1x penicillin/strepomycin and 1x fungizome + 100 g/ml Pm)(this will be sufficient for both lab partners)

40 mls 1x Spp___mls Pen/Strep stock___mls fungizome stock___ mls Pm stock

-Pipet 1.5 mls of 1x Spp medium + drugs into each of 24 wells -Select 12 drug resistant transformants for each construct.-Aliquot 30 l/well of each to individual wells in plate – Label each well.-Incubate plates in Tupperware boxes at 30oC for 1-2 days to allow transformants populations to grow.

27


------------------------------------------------------------------------Visualizing GFP localization------------------------------------------------------------------------

Overview of steps3. Add CdCl2 to cells to induce expression4. Visualize GFP expression

Materials:24 well tissue culture plates containing selected transformants Tissue Culture Plates (6 well)1xSPP medium200x fungizome stock1000x penicillin/streptomycin stock1 mg/ml CdCl2 stock10 mg/ml paromomycin (Pm) stock

Induce GFP expression from the Mtt1 promoter by addition of CdCl2 to 1.0 ug/ml to each culture of Tetrahymena

Prepare:1x Spp (+1x penicillin/strepomycin and 1x fungizome + 100 g/ml Pm +/- 1g/ml CdCl2)

___ mls 1x Spp___mls Pen/Strep stock___mls fungizome stock___mls CdCl2 stock (omit for one plate)___ mls Pm stock

Place 1.5 mls into selected wells of a 24 well plate, Select two of your transformants and inoculate these into two wells each. One well with be induced overnight and the other will be used for 1-2 hour induction. Some fluorescent protein fusions are very stable and can be visualized easily after overnight induction of expression, others are best visualized within a few hours of induction. Incubate at 30 degrees Celsius overnight to induce GFP expression.

To induce expression:At beginning of lab period, add 15 ul of 0.1 mg/ml CdCl2 ( a 1:10 dilution of your 1 mg/ml stock) to each well and mix thoroughly to induce GFP expression (for at least 1 hour at 300C). Visualize by Fluorescence microscopy.

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To check for GFP localization

Materials:Microscope slides and coverslips2% methylcellulose0.5 ml tubes10ug/ml DAPI

Spin down 200 ul of culture in tabletop microcentrifuge for 2-3 minutesquickly aspirate off excess mediumAdd 1 ul of DAPI solution and incubate at Room Temperature for at least 10 minutesplace 5 ul spot of 2% methylcellulose on slideadd 1-2 ul of concentrated cellsaffix coverslipvisualize fluorescence intensity and localization on microscope

29

Biology 3492 Spring 2008Protein Expression Module-------------------------------------------------------------------------------------------------Preparing whole-cell protein extracts for Western blot analysis -------------------------------------------------------------------------------------------------Overview of steps

1. Add CdCl2 to Spp cultures to 1 ug/ml. Induce expression 1-2 hours at 30oC.2. Harvest cells and lyse, store at -80oC.

For each of our constructs that we have electroporated into Tetrahymena, we will examine the protein both by GFP localization and by Western blot analysis. This analysis of the protein by SDS-Polyacrylamide gel electrophoresis (PAGE) will allow us to determine the amount of protein synthesized and what form it takes within the cell. To do this part, we need to extract the protein from the induced Tetrahymena .

Materials:6 well tissue culture plates containing transformants in 1xSPP medium +/- CdCl2

0.1 mg/ml CdCl2 stock15 ml conical tubesmicrofuge tubesWhole cell lysis buffer2x laemli sample buffer25x protease inhibitors1M dithiothreitol (DTT)

A. Induce the expression of the Mtt1-GFP fusion protein by addition of CdCl2 to a final concentration of 1 ug/ml to turn on transcription of the Mtt1 promoter. Incubate at 300C both overnight and for 1 to 2 hours (1.5 hours optimal) to allow for accumulation of the Tagged protein.

B. Prepare:0.5 mls of lysis buffer by adding 20 ul of protease inhibitor stock to bring to 1X (Place on ice until ready to use)0.5 mls of 2x Laemli sample buffer by adding 5 ul 1M DTT (leave at room temperature)

C. Whole cell protein lysis:Pipette 5 mls of induced cell culture into conical tube.Spin at full speed in Table top centrifuge for 2 to 3 minutesDecant (dump off quickly) growth mediumAdd 1ml of 10 mM Tris-HCl (pH 7.4) to resuspend and wash cells from mediumTransfer cells to labeled microfuge tubeSpin 2-3 minutes in benchtop microfugeAspirate of as much of the supernatent as possiblevortex gently to loosen cell pellet and place on iceAdd 50 ul of lysis buffer, mixing with pipet tip (avoid generating bubble though)Add 50 ul of laemli sample buffer, vortex briefly.Place cap lock on tube and boil for 10 minutesQuick freeze on dry ice.Store in ultralow freezer until ready to run on SDS- PAGE gel.

30

Biology 3492 Spring 2008Protein Expression Module-------------------------------------------------------------------------------------------------------------Western blot analysis of GFP-fusion proteins in whole cell extracts-------------------------------------------------------------------------------------------------------------Overview of steps

3. Add CdCl2 to Spp cultures to 1 ug/ml. Induce expression 1-2 hours at 30oC.4. Harvest cells and lyse, store at -80oC.5. Electrophorese samples on pre-cast 10% PAGE gel6. Transfer proteins to nitrocellulose membrane

Background:Examining GFP localization lets us see where our tagged protein localizes in the cell, but doesn’t fully let us evaluate the form (intact?) or the abundance of the protein. We can gain additional information about our protein expression by Western blot analysis. For each of our constructs that we have electroporated into Tetrahymena, we will isolate whole cell protein separate them from one another by size using SDS-Polyacrylamide gel electrophoresis (PAGE). The SDS (detergent) in the gel and lysis buffer coats the protein to give them a fairly uniform charge so that they will mostly be separated top to bottom = big to small proteins. We will detect our proteins using an antibody that recognizes GFP. This will allow us to determine the amount of protein synthesized and what form our fusion takes within the cell.

Materials:6 well tissue culture plates containing transformants in 1xSPP medium +/- CdCl2

0.1 mg/ml CdCl2 stock15 ml conical tubesmicrofuge tubesWhole cell lysis buffer2x laemli sample buffer25x protease inhibitors1M dithiothreitol (DTT)

A. Induce the expression of the Mtt1-GFP fusion protein by addition of CdCl2 to a final concentration of 1 ug/ml to turn on transcription of the Mtt1 promoter. Incubate at 300C both overnight and for 1 to 2 hours (1.5 hours optimal) to allow for accumulation of the Tagged protein.

B. Prepare:0.5 mls of lysis buffer by adding 20 ul of protease inhibitor stock to bring to 1X (Place on ice until ready to use)0.5 mls of 2x Laemli sample buffer by adding 5 ul 1M DTT (leave at room temperature)

C. Whole cell protein lysis:Pipette 5 mls of induced cell culture into conical tube.Spin at full speed in Table top centrifuge for 2 to 3 minutesDecant (dump off quickly) growth mediumAdd 1ml of 10 mM Tris-HCl (pH 7.4) to resuspend and wash cells from mediumTransfer cells to labeled microfuge tube

31

Biology 3492 Spring 2008Protein Expression ModuleSpin 2-3 minutes in benchtop microfugeAspirate of as much of the supernatent as possiblevortex gently to loosen cell pellet and place on iceAdd 50 ul of lysis buffer, mixing with pipet tip (avoid generating bubble though)Add 50 ul of laemli sample buffer, vortex briefly.Place cap lock on tube and boil for 10 minutesQuick freeze on dry ice.Store in ultralow freezer until ready to run on SDS- PAGE gel.

SDS Poly-Acrylamide Gel Electrophoresis of Protein

We will use pre-cast 10 % polyacrylamide gels. We will run duplicates, one gel to stain to determine protein loading amounts, a second to blot and detect our fusion proteins with anti-GFP specific antibodies.

Fill the tank with the Electrophoresis buffer.

Boil samples for 5 minutes, spin 30 sec to pellet insolubles, and place on ice

Load 12 ul each of your protein samples into 10 well gel with loading tips to the bottom of the gel well using loading guides.

Also load 7 ul of BIO-RAD precision plus protein standardsColors: Yellow (10 kD), green (37 kD), pink (25 and 75 kD), purple (150 kD), blue (15, 20, 50, 100, and 250 kD)

Connect the power pack to the apparatus, and electrophorese @ 125 Volts, ~1 hour. until dye front reaches bottom of gel.

To stain the gel, remove from gel cassette and wash in H2O 3 times for 5 minutes to remove SDS. Cover washed gel with BIO-SAFE straining solution. Allow protein to stain for 30-60 min, and wash it several time with H2O to destain. Store in H2O.

See next page for blotting of gel to nitrocellulose membrane.

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Biology 3492 Spring 2008Protein Expression ModuleSetting up a Western blot using semi-dry electroblotterMaterials:Semi-dry electroblot apparatus 6- 3mm Filter paper sheets cut to size of gelNitrocellulose membrane cut to size of gel (HANDLE w/ Forceps)Gel with separated proteinsTray with dIH2O for wetting membraneTray with Western blot transfer buffer for wetting filters

Steps after running gel:-Place the 2 sheets of 3mm paper pre-wet in transfer buffer on the bottom electrode plate. -Separate plates containing protein gel, cut top of gel off 0.5 cm above largest protein marker and discard this top “stacking gel”-Overlay gel with dry 3mm filter-Lift gel off of plate onto filter, wet in transfer buffer, and place on filters on top of blotter-Soak the nitrocellulose membrane in dI H2O water for 1-2 minutes (longer is fine). -Place the nitrocellulose membrane on the top of gel. -Place 3 other sheets of 3mm filter paper pre-wet in transfer buffer on top of membrane. -Use a clean tube (such as a pasteur pipette), and press on stack and roll out all air bubbles. Make sure all elements of the stack fit together well. This is the key of an efficient transfer.

Wipe all excess of buffer off the bottom plate, to avoid bridges of buffer that may forms between the two electrodes. (this is also critical to have a homogenous and efficient transfer). But, do not wipe too much either. Finally, place carefully the anode avoiding moving the stack.

This procedure should not exceed 15 min, because proteins diffuse within the gel and you could lose resolution by being to slow in your handling.

Attach semi-dry apparatus to power supply and select constant current option. Transfer time is calculated at 1 to 2 mA / cm2 gel area (for all gels) for 1.5 hours. Ready to begin detection.

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nitrocellulose membrane- wet in dH2O

Protein gel

3mm filter-wet w/ transfer buffer

3mm filter-wet w/ transfer buffer

electroblotter bottom

transfer direction

electroblotter lid

Biology 3492 Spring 2008Protein Expression ModuleDetecting specific proteins using antibodies

Background:Antibodies serve as specific reagents to detect proteins of interest. As we have tagged our MLH proteins with GFP protein sequence, we can detect our GFP-MLH fusion proteins by incubating our nitrocellulose filters with anti-GFP specific antibodies.

Materials:Plastic trays for washes1x into Phosphate-buffered saline (PBS) 1x PBS + 5% non-fat dry milk1x PBS + 0.1% tween 20 (a non-ionic detergent) +1% non-fat dry milk + diluted GFP antibodies1x PBS + 0.1% tween 20 (a non-ionic detergent) 1x PBS + Goat-anti-Rabbit, horse raddish peroxidase (HRP) conjugated secondary antibodiesHydrogen peroxide SuperSignal West Cheminluminescent SubstrateTwo plastic sheetsX-ray film

Tuesday;After blotting, place membrane into Phosphate-buffered saline (PBS) + 5% non-fat dry milk to block non-specific binding of antibodies (either 1 hr at room temp. or overnight at 4 oC).

Wednesday:Incubate the membrane overnight @ 4°C with the primary antibodies (Rabbit polyclonal anti-GFP antibodies for us) diluted in 1x PBS + 1% milk with gentle rocking.

Thursday:Wash 5 times 5 min in 1x PBS + 0.1 % Tween (15-25 ml each wash) at Room Temp, with gentle rocking.

Then, incubate the membrane 45 minutes to 1 hour at Room Temp with the Goat-anti-Rabbit, horse raddish peroxidase conjugated secondary antibodies in PBS and gentle rocking.

Wash 3 times 5 min in 1x PBS + 0.1 % Tween (15-25 ml each wash) at Room Temp, with gentle rocking.

Wash 2 times 5 min in 1x PBS (15-25 ml each wash) at Room Temp, with gentle rocking.

Mix equal volumes of hydrogen peroxide with SuperSignal West substrate (0.5 mls of each for 1 mini-gel) during last wash.

Remove from membrane wash buffer with forceps, drain off excess buffer, place on plastic sheet.

Overlay with mixture of chemiluminescent substrate, incubate 5 minutes at room temperature.

Detect signal using autoradiography film or fluorescence imager.Store in blot in PBS at 4 oC.

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Biology 3492 Spring 2008RNA Expression Module-------------------------------------------------------------------------------------------------------------rtPCR analysis of gene expression of isolated Tetrahymena RNA--------------------------------------------------------------------------------------------------------------

Overview of steps1. Isolate Total RNA from Tetrahymena (Thursday)2. Convert RNA to cDNA using reverse transcriptase (next Tuesday)3. Gel electrophoresis of products (next Thursday)

Background:There are several ways to determine the level of gene expression. Most rely on isolating RNA and determining the abundance of the steady-state level of messenger RNA. These methods include detection of gene specific RNAs by hybridization with labeled probes (i.e Northern Blot analysis) or enzymatic modification of sequence specific reagents (e.g. RNAse protection, S1 Nuclease digestion). We will use reverse transcriptase PCR, a very sensitive method, to monitor the expression of our predicted genes. rtPCR relies on the conversion of RNA to complementary DNA then using this DNA as template in specific PCR reactions. By varying the cycle number for the PCR, one can make the technique semi-quantitative. The conversion of mRNA into single-stranded cDNA for reverse transcription PCR is performed with the retroviral-derived RNA dependent, DNA polymerase enzyme called reverse transcriptase. Intact mRNA is first treated with DNAseI to remove any contaminating DNA. Then the RNA is hybridized to random hexamer primers (or oligo-dT primers in other cases) and copied by reverse transcriptase. The resulting product is suitable for PCR analysis of the converted RNA. We designed primers using the Primer3 http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi program to amplify a 200 to 300 bp region within each gene to monitor the levels of RNA in cells at various growth or developmental stages of Tetrahymena. These different stages will be vegetative growth, starvation, and time points during conjugation (3, 6, 9, and 12 hrs). Each team will isolate RNA from one stage and the material will be shared with the other teams to set up the cDNA synthesis reactions.

Tetrahymena genomic DNA

PCR primersPCR product

Dic gene coding region

35


Biology 3492 Spring 2008RNA Expression ModuleIsolation of Tetrahymena RNAMaterials:(Everything should be done under RNase-free conditions - see "RNase-free

methodology”)1. Tetrahymena cells in appropriate growth phase.2. RNA-Sol1

3. Cold chloroform with 1% isoamyl alcohol4. Cold isopropanol5. 50 and 15 ml conical collection tubes 6. RNAse-free microfuge tubes7. Clinical centrifuge

Procedure:1. Concentrate 15 mls of cells in conical tubes for 3 min at 2,500 rpm.2. Aspirate supernatant. 3. If cells are in growth medium resuspend pellet in 10 mM tris, pH 7.4; pellet.

spin cells again and discard supernatant4. place tubes in ice bucket.5. Resuspend pellet in small amount of leftover fluid (~60 ul/15 ml of cells).6 Add 9 volumes of RNA-sol (e.g. 600 l RNA-sol to 60 l pellet.). MIX by

pipetting up and down with p-1000.7. Transfer to clean microfuge tube pre-chilled in ice-bucket.8. Add 1/10 of the volume (to 60 l) in the tubes of chloroform-isoamyl alcohol.9. Seal tube well and vortex for 30 seconds. 10. Incubate for about 5 to 10 minutes on ice.11. Spin 3-5 minutes at full speed in 4oC microfuge12. Remove the aqueous layer to fresh, pre-chilled tube.13. Extract with equal volume (~350 l) of chloroform/isoamyl alcohol14. Add an equal volume of isopropanol. Keep tube on ice.

15. Store at -20o C if not using.

1This can be bought or made up. To make up, combine: 1 part H2O-saturated phenol 1 part of "solution B": 4M guanidinium thiocyanate + 25 mM sodium citrate (pH 7.0). 0.1 part 3M NaOAc Supplement with 340 l -mercaptoethanol per 100-ml of total solution Store at 4o C; can be kept for a few months

36

Biology 3492 Spring 2008RNA Expression Module

PROCESSING OF RNA (POST-ISOPROPANOL) Materials:1. Cold 70% ethanol made up with DEPC-treated H2O.

2. RNAse-free DEPC-treated H202

3. RNAse-free 1.5 ml Eppendorf tubes

Precipitation of RNA from isopropanol1. Remove 200 l sample from cold storage in isopropanol, and distribute to 1.5-

ml eppendorf vials2. Centrifuge in cold at full speed for 8-10 min.3. Remove supernatant4. Add cold 70% ethanol made up with DEPC-H2O; (5. Repeat ethanol wash for

large pellets).6. Air Dry or dry under vacuum for 5-10 minutes.

7. Resuspend in 25 l DEPC-treated ddH2O

Determination of RNA concentration1. Turn on UV lamp of spectrophotometer to warm-up. Make 1:1000 dilution of

RNA in sterile H2O2. Calibrate spectrophotometer at 260 nm using 400 l of Sterile H2O in quartz

cuvette3. Measure O.D.260nm of diluted RNA4. Calculate RNA concentration of starting RNA solution (dilution factor X 40

g/ml per O.D.260nm unit)

2 DEPC Treatment inactivates RNAses: Add DEPC to ddH2O to make up a 0.1% solution; shake vigorously, then let stand for at least 12 hr; finally autoclave for at least 15 min (one can also boil off the DEPC).

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------------------------------------------------------------------------

BASIC PROTOCOL: CONVERSION OF mRNA INTO Single-Stranded cDNA

Materials Tetrahymena RNA resuspended in RNAse-free H2O10 X DNAseI bufferDNAseI enzyme10 X DNAseI stop buffer (20 mM EGTA)37oC incubator65 oC water bath

10 mM 4dNTP mix 5 X reverse transcriptase (RT) buffer (see enzyme sheet) 0.1 M dithiothreitol (DTT) 3 mg/ml random-hexamer primers RNase out ribonuclease inhibitor (Invitrogen; stored at –20C) Superscript II reverse transcriptase (Invitrogen) DNAse I treatment of RNAset up 20 l reaction with: 15 g of RNA ___ l1 X DNAseI buffer ___ l2 units DNAseI (1 unit/ l) ___ l20 units RNAse out (40 unit/ l) ___ l

Incubate 30 minutes at 37 0C

Add 2.25 l of DNAseI stop bufferHeat at 65oC to inactivate DNAseI for 10-15 minutesHeat to 90oC and place on ice

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Synthesize cDNA

Prepare cDNA synthesis reactions for vegetative cell RNA, Starved Cell RNA, 3, 6, 9, and 12 hour mating cell RNA (early to late development. Prepare duplicates for each, omitting reverse transcriptase from one, for total of eight reactions. Figure out reverse transcription reaction volumes and write them in your notebook BEFORE next Thursday’s lab period

In separate PCR tubes on ice, add in the following (25 l total): 13 X Master mix

aliquot 21 l/tube 1.25 l 10 mM dNTPs (500 uM final each) ___ l 5 l 5 X 1st strand synthesis buffer (1X final) ___ l2.5 l 0.1 M DTT (10 mM final) ___ l0.5 l 3 mg/ml random hexamers ___ l10.75 l H2O to 25 l ___ l 1 l (40 U) Rnase out ribonuclease inhibitor ___ l

then to each tube add the appropriate RNA and enzyme 3 l DNAse treated RNA (2 g) 1 l Superscript II reverse transcriptase

(or omit for –RT reactions)

Incubate in PCR machine at 42 oC for 50 minutes, 85 oC for 5 minutes. Proceed to PCR reaction or Store at –20 oC

39

Biology 3492 Spring 2008RNA Expression Module--------------------------------------------------------------------------------------------------PCR Reaction conditions

We will use our gene specific primers that we designed using PRIMER3 for our rtPCR. We will also include in the reactions primers for the alpha tubulin gene (TubA) which is highly expressed at all times during Tetrahymena growth and development. This gene will serve as a control that the cDNA synthesis was efficient. We will set up reactions that will run for 28 or 34 PCR amplification cycles to allow us to semi-quantitatively measure relative amounts of expression in each sample (34 cycles will amplify less abundant RNAs). In addition to the twelve cDNA reactions (+ Reverse Transcriptase) we will do a duplicate at 34 cycles using our –RT control reactions. Also, to control that the PCR worked, we will use genomic DNA as a control for the PCR amplification. (NOTE: While this is the best way to get some quantitative measure, we will first do a single 32 cycle PCR -- +/- RT rather that the two different cycle number) therefore you will have 18 total rtPCR reactions +1 genomic DNA (28 cycle only). Figure out PCR reaction volumes for each primer set and write them in your notebook BEFORE next Tuesday’s lab period.

Set up reactions on ice with everything except for cDNA genomic DNA template.

Reaction components Amount for 1 rxn ( l) 15 rxn master mix 10x PCR buffer25mM MgCl2 (use 1 l/reaction)10 M rtPCR forward Oligo (use 1 l/reaction)10 M rtPCR reverse Oligo (use 1 l/reaction)10 M TubA604 Oligo (use 1 l/reaction)10 M TubA975r reverse Oligo (use 1 l/reaction)100x dNTPs (10mM)Taq DNA polymerase (use 0.3 ul/reaction)H20 to 30 lcDNA or Genomic DNA (use 1 l/reaction) (not in master mix) Total 30 l

PCR cycling conditions:1 cycle 94oC for 4 min

32 94oC for 30 seccycles 56oC for 30 sec

72oC for 50 sec

1 cycle 72oC for 5 min.

Pour 2% agarose gel and electrophoresis products

40

Biology 3492 Spring 2008RNA Expression ModuleRNase free PRECAUTIONS--Wear Gloves at all times--Keep RNA on ice at all times--Treatment of glassware and other materials:1. Chromic-acid wash (rinse well!) and bake glassware (240o C) 5 hrs to overnight

2. Use brand new plasticware3. Soak stir bars, bottle caps, and other non-bakeable articles overnight (12 hr or

more) in 0.1% DEPC (diethylpyrocarbonate) in ddH2O. Then boil for 15 min. or more (or autoclave) to break down the DEPC. These can be stored in an acid-washed, baked beaker.

All solutions except Tris buffers:1. Make up the solution using acid-washed baked glassware, RNase-free stir bar,

baked spatulas, gloves, using ddH2O.

2. Add DEPC to 0.1%; let sit at least 12 hr at room temperature.3. Autoclave at least 15 min.

Tris-containing solutions1. Make up using acid-washed, baked glassware, RNase free stir bas, no

spatula, gloves, and DEPC treated and autoclaved ddH20.

2. Autoclave

Gel boxes and combs1. Rinse for about 30 min with 1% SDS in 0.5 N NaOH.2. Rinse with dH2O

3. Dry with methyl (or ethyl) alcohol and kimwipes.

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Biology 3492 Spring 2008Appendix 1: Sample syllabus

Biology 3492: Laboratory experiments with eukaryotic microbesSpring 2008 (subject to change)

Jan 15 Computer lab (NSLC): Introduction to the Tetrahymena Genome Database (TGD)Jan 16 Lecture 1: Scientific approaches using model systemsJan 17 Computer lab: Predicting coding regions for analysis

Jan 22 Working with microorganisms; aseptic technique and cell counting (gene map due)Jan 23 Lecture 2: DNA manipulations Jan 24 Microscopes: making an invisible world visible; PCR amplification of candidate genes

Jan 29 Gel electrophoresis of PCR reactions; TA cloning and E. coli transformation Jan 30 Lecture 3: The cytoskeleton and the Tetrahymena membrane skeleton (Lab report 1 due)Jan 31 PCR screening of E. coli transformants

Feb 5 Plasmid DNA isolation, Restriction enzyme analysis, DNA sequencing Feb 6 Lecture 4: DNA transformation techniquesFeb 7 Fusion of gene of interest to Fluorescent protein sequence

Feb 12 Plasmid DNA isolation, Verification of correct fusion by restriction enzyme analysisFeb 13 Lecture 5: Microscopy techniques Feb 14 Tetrahymena electroporation

Homework: Selection of Transformants arrange time with Prof. Chalker

Feb 19 Scoring Tetrahymena electroporation; cultures for microscopyFeb 20 Lecture 6: The Tetrahymena membrane skeletonFeb 21 Fluorescence microscopy of GFP-MLH fusions

Feb 26 Fluorescence microscopy of GFP-MLH fusions; Protein isolationsFeb 27 Lecture 7: Genetic AnalysesFeb 28 Fluorescence microscopy of GFP-MLH fusions; Protein isolations

Mar 4 Western-blot analysis of GFP-fusions; RNA isolationsMar 5 Discussion: Giving Scientific Presentations: (Lab report 2:draft 1 due)Mar 6 Western-blot analysis of GFP-fusionsSPRING BREAKMar 18 Oral presentations of protein localization (Notebooks Collected and Graded)Mar 19 Lecture 8: Assessing protein:protein interactionsMar 20 Oral presentations of protein localization (Notebooks Collected and Graded) Mar 25 rtPCR expression analysis (Lab report 2 due)Mar 26 TBA Mar 27 rtPCR expression analysis (continued)

Apr 1 Gel electrophoresis of rtPCR analysis; Begin co-localization or gene knockdown expts.Apr 2 In Term ExamApr 3 Co-localization or gene knockdown expts

Apr 8 Co-localization or gene knockdown exptsApr 9 TBAApr 10 Co-localization or gene knockdown expts

Apr 15 Co-localization or gene knockdown exptsApr 16 TBA Draft of final report dueApr 17 Co-localization or gene knockdown expts

Apr 22 Wrap-up

Apr 23 Discussion of DataApr 24 Wrap-upApril 28 – undergraduate research symposium(Final Report due Thursday, May 1)TBA= to be announced

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Biology 3492: Laboratory experiments with eukaryotic microbes

Spring 2008Description:Laboratory Experiments with Eukaryotic Microbes. An introduction to diverse molecular and cell biology techniques used in model experimental organisms to explore fundamental biological questions. Experiments will be performed using selected fungi and protozoans commonly used in major research efforts. Emphasis will be placed on choosing the appropriate organism for the question posed using the most current technologies. Prerequisites: Bio 2960 and 2970 and permission of instructor. One hour of lecture and six hours of laboratory a week. This course fulfills the laboratory requirement for the Biology major. Enrollment limited to 12.

Meeting Times:Laboratory: Tues/Thurs 9 am – 12 noon; in Rebstock 126Lecture: Wed 3 pm -- 4 pm; in Life Sci. 118

Instructors:Professor Douglas Chalker. (coursemaster): 935-8838; [email protected]

Monsanto Hall 304, Hilltop

Monica Sentmanat (TA): emailSidney Wang (TA): email

Grading:Lab Notebooks

Midterm evaluation 75 ptsFinal evaluation 75 pts

Lab write-up 1 100 ptsLab write-up 2 150 ptsLab write-up 3 (Complete project) 150 ptsOral presentation 1 100 ptsPoster 100 ptsPoster presentation (Saturday, April 26th) 100 ptsIn-term exam 150 ptsProblem sets/exercises 100 pts Total 1100 pts

General Policies:You are expected to attend every lab and lecture session. This is a laboratory course, so hands-on experience you will gain has the major instructional value. Arrive on time so that we can complete the experiments planned. You will be working as a team this semester, so those arriving late affect everyone. If you know you have to miss a class, please inform both me and your partner. We have only one exam, so make sure that you do not miss this class period as no make-up exam will be given. A doctor's note stating that you were seen for an illness of sufficient severity to warrant an excuse is needed - a note simply stating that you visited the health center is insufficient. In the event of a death or serious illness in the family, certification will be needed to validate your absence. If you have a legitimate excuse for an absence, your final grade will be determined by calculating the mean of the other assignments. Unexcused, missed assignments will be given a grade of zero and may well necessitate withdrawal from the course.

Lab Reports

You will have three lab reports due this semester. Some will require drafts prior to the final report. The final report is a cumulative report of all your lab work this semester. These will take the form of a scientific paper. The due dates on the syllabus are tentative, depending upon our progress with our experiments.

Lab Notebooks

You will need to purchase a bound lab book for the semester. Keep all your notes and experimental procedures in this book. This will need to be left with Prof. Chalker at the end of the semester, but you are welcome to photocopy your notebook (I will give you access to a copier if you wish to do this).

Plagiarism

Definition (from www.Dictionary.com): n 1: a piece of writing that has been copied from someone else and is presented as being your own work 2: the act of plagiarizing; taking someone's words or ideas as if they were your own.Plagiarism will be taken very seriously and will be reported to the dean’s office for appropriate action. In writing assignments, be careful not to simply copy reference material, but use it to help you formulate and support your own thoughts and ideas. Always give proper reference to material used. Long sections of text taken verbatim should always be in quotations, but try to avoid using this style in most scientific writing. Make your own conclusions, don’t just rely on what you read.For those using the Credit/No Credit option, a grade of C- is required to receive credit.

Biology 3492 Spring 2008Appendix 2: Working with Microbes Intro Lab

------------------------------------------------------------------------WORKING WITH MICROORGANISMS ------------------------------------------------------------------------In the best of their possible worlds, bacteria can reproduce until they reach population densities of approximately 109 (one billion) /ml. Single-cell eukaryotes can reach densities of 108/ml. Thus, it becomes necessary to dilute them in order to isolate single organisms, estimate their numbers, or prepare smaller populations for analyses. When cells are suspended in liquid, they are diluted by mixing a measured volume of the culture with measured volumes of sterile water, buffer, or broth. Because the ability to work with dilutions is so important to microbiology, we will learn how to do this in practice.

Most of the exercises and experiments you will perform during this semester use sterile media and pure cultures (progeny of a single organism). A series of operations have been developed to limit the risk of contaminating these materials during manipulations. These operations are known as aseptic techniques. They also help protect investigators from infecting themselves or releasing the organisms into the environment. Learn the following rules and procedures and understand how each requirement contributes to maintaining asepsis.

General Rules

1. Work on a clear tabletop. Put all unnecessary items away.

2. Wear a lab coat; wash hands before performing any manipulations and after you are through.

3. Disinfect the benchtop with an appropriate disinfectant before you begin working and after you are through.

4. Keep all cultures closed and tubes upright in a rack until ready for use.

5. Work quickly without disturbances.------------------------------------------------------------------------

Aseptic Technique for Preparation of Agar Plates

1. Melt sterile agar and place the container in a water bath at 45-50°C. Make sure that there is enough water to cover the agar. If necessary, use a lead "doughnut" to prevent the agar-containing vessel from tipping.

2. Place sterile petri plates on a disinfected benchtop.

3. Remove the lid from the agar-containing vessel and pass the mouth through a flame to destroy any contaminating organisms. Hold the container at an angle and not vertical.

4. Pour the agar into the petri dishes. Gently swirl the plate to distribute the agar to cover the bottom of the plate.

5. If any agar remains in the container, pass the mouth through a flame and close the vessel. Return it immediately to the water bath.

6. Allow the plates to solidify. Label the plates with the type of media they contain and the date they were poured.


7. If plates are being stored for more than 48 hours, seal them in a plastic bag.------------------------------------------------------------------------

Aseptic Technique for Transfer of Organisms

Removal from Broth Cultures or Solid Media a. Microbes can be removed from broth with an inoculating loop or a pipette; they can be removed from solid media with an inoculating loop, needle, or sterile toothpick.

b. Inoculating loops and needles are made from metal wire and can be sterilized by flaming until red-hot. The entire length of the metal wire must be sterilized. This is done by flaming from the handle end to the tip (avoiding aerosolizing liquid in the loop). Flaming incinerates all organisms that are present. Once sterile, the loop is inserted into a tube of broth and liquid is lifted out.

The needle is touched to the region of growth on the solid medium.

c. Pipettes are either purchased sterile and disposable or are sterilized in cannisters prior to use. Remember to keep the pipette cannister on its side. DO NOT STAND IT UP. (Why?) When lifting a pipette from the cannister, touch only at the mouthpiece and only one pipette. Close the cannister when you have removed the pipette. Never return pipettes to the cannister. A pipettor is always used to remove a portion of the broth. Handle only the upper portion of the pipette when inserting it into the pipettor. If the tip should inadvertantly touch another surface, do not use the pipette. Take a new one.

Micropipettors with sterile tips can also be used.

1. Transfer to Broth Media in Flasks or Tubes

a. After the lid is removed, flame the mouth of the flask or tube.

b. Holding the vessel at an angle, introduce the loop or needle into the liquid and agitate gently. If a pipette is used, a measured volume of liquid can be released using the pipettor.

c. Flame the mouth of the flask or tube before returning the cover. Flame the loop or needle after use to incinerate any remaining organisms. Dispose of all contaminated pipettes or tips in the appropriate containers.

2. Transfer to Agar Plates

A. SPREAD PLATE1. Pour a plate and allow to solidify.

2. Suspend the organisms in the broth culture. Remove the cap from the culture tube and flame the mouth of the tube. Do not contaminate the cap during this procedure. Remove 0.1 ml of organisms. Flame the mouth again and cover the tube.

3. Place the organisms in the center of the plate.

4. Dip the bent glass rod in the alcohol and shake off excess liquid. Keep the alcohol container away from the flames.

Biology 3492 Spring 2008Appendix 2: Working with Microbes Intro Lab5. Carefully flame the rod. When all the alcohol has burned, allow to cool for a few seconds. You may be sure the rod is cooled by placing it on the agar surface at the edge of the plate.

6. Use the rod to spread the organisms over the surface of the plate while rotating the plate on the desktop.

7. Return the glass spreader to the alcohol.

8. Incubate the plate for overnight to 72 hours (depending upon organism plated) in an inverted position. Look for isolated colonies. -----------------------------------------------------------------------B. QUADRANT STREAK PLATE1. Pour a plate of TSA and allow to solidify. Return the TSA to the water bath.

2. Suspend the organisms in the broth culture or use directly from a slant. Flame the loop and wire until it is red hot. Remove the cap from the culture tube and flame the mouth of the tube. Do not contaminate the cap or the loop during this procedure. Remove a loopful of organisms. Flame the mouth again and replace the cap on the tube.

3. Spread the organism over a small region on the edge of the plate as in 1 in the diagram below.

4. Flame the loop and let it cool for a few seconds.

5. Streak from the end of region 1 across the edge of the plate forming region 2.


7. Streak from the end of region 2 across a quarter of the plate forming region 3.


9. Streak from region 3 across the remaining portion of the plate forming region 4.

10. Flame the loop before setting it down.

11. Incubate the plate for overnight to 72 hours (depending upon organism plated) in an inverted position. Look for isolated colonies.

PIPETTING

A small volume of cells is withdrawn from the culture using a sterile pipette. Be sure to handle only the upper portion of the pipette. Do not touch the tip or it will no longer be sterile. A bulb or pipette aspirator is used to draw fluid into the pipette. Pipetting should never be performed by mouth. Two types of pipettes are used routinely in microbiology laboratories. They are the serological (blow-out) and measuring (delivery) pipettes. The serological pipette is calibrated from the tip to the zero mark. The entire volume is delivered by emptying the pipette. The measuring pipette is calibrated from a mark above the tip to the zero mark. After the measured amount of liquid is released, the flow is stopped. Both pipettes can be used to deliver less liquid than the total volume of the pipette. It is important to know which type of pipette you are using so that you do not deliver an incorrect volume.

Biology 3492 Spring 2008Appendix 2: Working with Microbes Intro LabMicro pipettors (“pipetmen”) with sterile disposable tips can also be used to deliver volumes ranging from 1 ul to 5 ml.

You should practice pipetting and determine your precision, how consistent are you in delivering the same amount each time. This can be done by using the same pipette to repeatedly deliver a fixed volume of water. The water is weighed each time to determine the amount delivered. Do this 9 times using a nonsterile 1 ml pipette and dispensing 0.5 ml each time.

The accuracy of the pipette is defined as how close the volume delivered is to the actual volume indicated by the pipette markings. You can get an idea of accuracy by comparing your measurements with those of other class members. How much do they vary?

DILUTING

Dilutions are generally performed in multiples of two or ten. This makes it easier to perform and to calculate concentrations. For example, a 1 to 10 dilution (written 1:10) is 1 volume of bacteria into a total of 10 volumes of liquid. This means that one volume of bacteria is added to 9 volumes of diluting liquid (making a total of 10 volumes).

How would you dilute a broth culture containing 100,000 organisms/ml to make one containing 100 organisms/ml ? You could take 1 ml of the culture and place it into 999 ml of water. For reasons of practicality and accuracy, dilutions of large numbers of bacteria utilize a serial dilution, series of smaller dilutions rather than one large dilution. The final dilution is calculated by multiplying each of the smaller dilutions. Thus, if 1 ml of culture is diluted into 9 ml of water (1:10) and 1 ml of this diluted material is added to another 9 ml of water (1:10), the final dilution will be 1:10 x 1:10 or 1:100. A third 1:10 dilution would give a final dilution of 1:1000.

Of course, to know the actual cell number, one has to count them. To determine the actual number of organisms in each tube, a 0.1 ml sample can be removed and plated. Colonies, each one derived from a single cell, can be counted to determine cell number in starting culture.--------------------------------------------------------------------------------------------------------------------

Biology 3492 Spring 2008Appendix 2: Working with Microbes Intro LabCOUNTING CELLS USING A HEMOCYTOMETER.

In addition to using plating techniques to determine cell numbers we will use manual cell counting (for both yeast and Tetrahymena). Although there are now automatic cell counters, they are quite expensive and can break. Manual cell counting is still an extremely useful technique. While today’s exercise introduces you to cell counting techniques, more importantly, it hopefully will give you a feel for important experimental variables, such as sampling errors, and will require that you be attentive to seemingly trivial aspects of experimental technique that often contribute to the reproducibility of a result. Note that you will have to count two different types of cells.

The hemacytometer

The principle behind microscopic cell counting is quite simple: a known volume of cell culture is counted and the total number of cells (usually in a ml) is calculated. The device we will use is called a hemacytometer because it is most often used to count blood cells. It has various rulings as shown below. The simplest dimension for us to use is the 1mm square. When a special cover-slip is placed over the hemacytometer, the height of the chamber is 0.1 mm. Thus, the volume of one of the numbered squares is 0.1 mm3 (0.1 µl) and the average number of cells in one square must be multiplied by 104 and by a factor to correct for the dilution (if any) to calculate cells/ml. Note that hemacytometers are precision instruments. They are expensive (>$50 apiece) and are fragile. They are inaccurate if dirty and must be used only with special cover-slips.

Motile cells (such as Tetrahymena) must be killed before counting (yeast cells can be counted directly). Your cultures of Tetrahymena should be at a reasonable density for counting directly but you will investigate for yourself the effects of dilution and concentration. Because they are large, all pipetting of Tetrahymena must be done rapidly from well suspended cultures to prevent them from settling out and causing sampling errors.

Although simple in principle, it often takes some practice to learn to fill a hemacytometer correctly. You must pay attention to internal consistencies (or lack thereof) that indicate that you are filling it properly. For example, you should have consistent readings from both sides of the hemacytometer and there should not be major discrepancies between the right and left squares or the top and bottom squares of a single side. Both partners should get similar results, although it is not unusual for different investigators to have small, consistent differences in their results based on small consistent differences in technique.

1) Place the clean and dry cover-slip on the clean and dry hemacytometer.

2) Load both sides of the hemacytometer with a pipetman. (Use 1/100 dilution of yeast culture and undiluted tetrahymena mixed 1:1 with 2% formaldehyde). Be sure that cells are well suspended and don't have time to settle out. Also be sure that the hemacytometer is filled just to completion. If the cover-slip is floating or if there is an air bubble visible you must start over. Both sides of the hemacytometer should be filled independently (i.e. not with the same pipet-full) SEE HANDOUT.

3) Count cells in 1 mm squares. Count at least the 4 corner squares on each side of the hemacytometer and count a total of at least 100 particles (if the sample is too concentrated, dilute it).

4) Compute the number of cells per ml

Biology 3492 Spring 2008Appendix 2: Working with Microbes Intro Lab SPECTROPHOTOMETRIC DETERMINATION OF CELL DENSITY.

Microorganism cell density is often measured using a spectrophotometer operated in the visible range. By checking the absorbance of a culture, one can estimate the cell number once a standard curve has been determined. Typically the density of baker’s yeast (Saccharomyces cerevisiae is measured at O.D. 600nm and log phase cultures range between 1x106 and 1x107 per ml. Tetrahymena are measured at 540 nm and log phase cultures range between 1x105 and 1x106 per ml. The instrument must first be calibrated with medium without cells (a blank) and then the culture density can be determined. Use your cell counts to determine how many cells in a give culture with and O.D. =X. Share your data with others at your table for your lab write-up.

---------------------------------------------------------------------------------------------------------------------LAB EXERCISE: RELATING CELL SIZE, DENSITY, AND NUMBER.

Materials:YPD culture medium for YeastSPP culture medium for TetrahymenaYPD plates for yeastCulture tubes for serial dilutionsHemocytometerCompound microscope

For today’s exercise, we will use two methods to determine cell number. One is direct counting using a hemocytometer. We will use this both for yeast and Tetrahymena. The other is plating yeast cells onto agar plates. For both, we will first need to dilute the cultures to get the cell density to a countable level. To dilute cultures, we will make serial dilutions, a method to accurately dilute cultures. We will dilute our yeast cultures in ten fold dilutions ranging to 1:10,000.

1. Determine the optical density of each culture using the spectrophotometer. Use 600nm for yeast, 540nm for Tetrahymena. Zero the spectrophotometer with a medium blank. Place 3 mls of culture in tube, and record the optical density.

2.Place 3 mls of YPD in each of 5 culture. To make the first 10 fold dilution, add 0.33 mls of the yeast culture to the first tube. To make the 1:100 dilution, take 0.33 mls from 1:10 dilution and add to the second tube. Repeat for all tubes to make a series of 1:10 to 1:10,000 dilutions. You will also need to make a 1:5000 dilution (how?).

3. After making dilutions, plate out 0.1 mls of the 1:5000 and 1:10,000 dilutions onto two YPD plates. After plates dry, incubate at 30oC until Thursday.

4. Count cells using hemocytometer. start with 1:1000 dilution, if too dense, move to lower dilution. For Tetrahymena, mix culture with an equal about of 2% paraformaldehyde. Count cells on hemocytometer.

5. Record all your data. Plot cell number as function of optical density

Lab write-up #1 (100 pts)

Cell size, density, and number correlation.

In this lab report, you should summarize your findings based on the second week of lab. Your report should include data tables of your cell counts and spectrophotometer readings for both yeast (Saccharomyces cerevisiae) and Tetrahymena thermophila (no common name). You should then plot these data on a graph to illustrate the relationship between cell density (optical density) and cell number for each. You should also have a table indicating estimated cell size for each yeast, Tetrahymena, and Chlammydomonas, Add a short description of the morphology of each.

In writing your report you will want to have the following sections:Introduction: In the introduction, go to the internet (use reliable sources) or the library and find information on each of these organisms. Write an introduction of one or two paragraphs mostly focusing on the features of these different microorganisms. I’m not asking for a long report on each, just a sentence or two about each that shows that you know a little more about each than we told you in lab. (Be sure to reference any sources).

Methods: Here describe what you did. How did you determine optical density and cell numbers? How did you do your dilutions? This should be very concise, but have enough information that your lab partners could repeat your experiments.

Results and Discussion: This is where you present the summary of your results in tables and graph forms. All Tables and graphs need to have a concise legend with a title. Don’t just show the tables, but describe what is in them. Discuss any consistencies and inconsistencies in your data and why you think they arose. I would probably first present and discuss the table comparing cell sizes. While we did that last, your presentation will likely flow better if you present it first, but this is up to you and the way your choose to present your data. For the cell counting exercises, compare the results between the yeast and Tetrahymena data. For example, why are the cell numbers for Tetrahymena different from yeast for the similar optical densities? etc.?

Lab report is due in lab on Thursday, Jan. 31, 2008.

Biology 3492 Spring 2008Appendix 3: Assignments, problems sets, and grading sheets

---------------------------------------------------------------------------------------------------------------Problem set 1: Verifying your pENTR_TopoD clone: Making a plasmid map (25 pts)---------------------------------------------------------------------------------------------------------------Objective: learn to use the GCK program to analyze sequences to aid in the design of investigations involving those sequences. A graphic representation of a DNA sequence is an important part of the design of experiments in molecular biology. One needs to be familiar with the overall structure/layout/features of the DNA of interest to enable future studies. You downloaded the sequence of your Tetrahymena gene of interest and can download the GCK sequence file of the vector pENTR_TopoD from the biol3492 server.

Assignment:1. Annotating your gene sequence.

a. Starting with your GCK Tetrahymena gene sequence file, Mark the following restriction sites on your sequence: ApaI, BamHI, BglII, BsrGI, EcoRI, EcoRV, HincII, HindIII, NotI, PstI, SacI, SalI, ScaI, XbaI, XhoIb. Make a separate file of your PCR fragment by copying the sequence from the region that your team is working with, then......c. add the extra sequence that you added to the 5’ ends of each oligonucleotide used in PCR and remark your sequences to see if any sites were added.

2. Combine your gene sequence with the already annotated pENTR_TopoD. e. Paste in the your expected PCR fragment sequence into the appropriate site in this vector

(marked insertion site: between position 688 and 689). Leave off CACC as this sequence recombines with the copy of this sequence in the vector. Save your files and copy to the server.

f. Print a list the positions of the following restriction sites: ApaI, BamHI, BglII, BsrGI, EcoRI, EcoRV, HincII, HindIII, NotI, PstI, Saci, SalI, ScaI, XbaI, XhoI.

g. Determine the exact predicted size of the restriction fragments from a BsrGI, an EcoRV, and a HincII digest.

**For all sequences, print out a display (print sequence of your gene sequences and graphical display of your pENTR file) of your genes and construct and copy files to Bio3492 server on Doug Chalker’s iMac. Turn in your printouts on Wednesday, Feb 7 in class.

MAKE SURE TO SAVE YOUR WORKAccessing Bio3492 file dropbox on Doug Chalker’s iMac in NSLC (to use to turn in computer files):In ‘Finder’ select ‘GO’:’Connect to Server’Select ‘Local’ folder: Select ‘Doug Chalker’s iMAC’:’Connect’: logon as ‘biol3492’ password: TthermophilaSelect ‘Public’ folder, select ‘dropbox’, select ‘Biol3492 seq files”; Find your gene sequence GCK file. Create a folder of your files on your computer’s desktop; before ending, be sure to Click and drag your folder into for future use dropbox.

Biology 3492 Spring 2008Appendix 3: Assignments, problems sets, and grading sheetsSome GCK function commands you will need:Make codon table for TetrahymenaOpen file; Construct: features :edit codon table: new codon table,Change TAA and TAG stop codons to Gln (glutamine); name ‘Tetrahymena’ ; ‘OK’

Select codon table for TetrahymenaOpen file; Construct: features :select codon table: Select ‘Tetrahymena’ ; ‘OK’

To show numbered coordinates of the sequenceOpen file Construct: Display: Show Positions

To group sequence for friendly displaySelect sequence; Format:Grouping:(select ‘by threes’ for protein sequence, ‘by tens’ for others)

To color a block of sequenceSelect sequence; Format:color:(select color)

To designate a regionSelect sequence; Construct: features :Make region

type in name, designate as protein if desired

To change regions arrow display to line or different arrowSelect region; Format: lines: (select line type)

To mark restriction sites deselect all sequence; Construct: features :mark sites

in popup window, select enzyme names one at time and ‘add’ to list, click ‘OK’

To mark a particular position (e.g. to indicate an oligo position)Select cursor location at site; Construct: features: mark location

To insert a particular sequence into an existing file (e.g. to indicate an oligo position)Select sequence to be inserted and copyPlace cursor over at site to insert sequence and paste sequence to this location Notes: By highlight a region of sequence in target file, the highlighted sequences will be removed during operationSequence can be inverted by ‘special paste’

To space restriction site markersSelect sites in graphic display Format:Sitemarkers:automatic arrangement (or just drag individually)

To generate list of restriction sites in your sequencedeselect any sequence; Construct: features :list sitesin popup window, select enzyme names one at time (or ‘add all’ and ‘add’ to list, click ‘OK’


insertion site

Biology 3492 Spring 2008Appendix 3: Assignments, problems sets, and grading sheets---------------------------------------------------------------------------------------------------------------Problem set 2: pENTR to pICY-gtw swap using LR recombinase: plasmid map (15 pts)---------------------------------------------------------------------------------------------------------------Assignment:1. Remove gateway cassette (in black in the GCK file) and replace with your gene sequence from your pENTR-.... with your gene of interest (GOI).

a. Using the diagram below, find sequences from your pENTR vector to swap with the gateway cassette in pIGY-gtw.

2. Determine the fragment sizes for diagnositic restriction enzyme digestsh. Determine the fragment sizes for pICY-gtw and your pICY-GOI cut with BamHI by

selecting all sites and copying to an new “gel” file.i. Determine the fragment sizes for pICY-gtw and your pICY-GOI cut with BsrGI, EcoRV,

and PstI by again selecting all sites and copying to the new “gel” file. j. Select one of these other enzymes to perform your diagnostic digests.


Print out a graphical display of your pICY-GOI map. Make sure that you display the construct title. Circle the enzyme cut sites for BamHI and the other enzyme of your choosing. Print out the table of fragment sizes for pICY-gtw and your new pICY-GOI cut with BamHI and also for the two plasmids cut with the enzyme of your choosing. Save the file of your construct and copy your new file to the Bio3492 folder on the NSLC server. Turn in a hard copy at beginning of class on Thursday, Feb 14, 2008.


QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

AttR1

AttR

YFP

MTT1 Promoter


insertion site

Attl1

AttR2Attl1AttR2

Biology 3492 Spring 2008Appendix 3: Assignments, problems sets, and grading sheets---------------------------------------------------------------------------------------------------------------Problem set 3: Verifying the sequence of your PCR clone in pICY-gtw (25 pts)---------------------------------------------------------------------------------------------------------------Assignment:Compare the sequence of your forward and reverse sequencing reactions to your predicted YFP fusion sequence in pICY-GOI clone.

1. Use the Align program to compare your sequences to your pICY-GOI sequence k. Copy sequence from ~50 bp upstream to ~50 bp downstream of your gene’s sequence,

paste into top dialog box of align program.l. Open your chromatogram file in FinchTV for your forward (upstream) reaction (the

forward reaction has mtt in the file name), Select the information icon to retrieve the sequence; copy and paste into bottom dialog box of align program. Run align.

m. Copy and paste your results into a word file (you will need to change the font size to 8 or 9 pt to preserve alignment.

n. Repeat steps b and c with your reverse reaction (the reverse sequence has cfg in the file name). You will need to reverse the orientation of the sequence before downloading the sequence information.


2. Working with the alignments and your chromatograms, verify or correct any mismatches a. Does your chromatogram confirm aligned mismatches, suggest incorrect base-calling by

the sequencing program, or leave you unsure? For each mismatch in your alignment, mark as either verified mismatch (M), correct sequence (C), Ambiguous (A) placing the letter above the position in the alignment.

b. Draw a diagram of your gene with a scale bar and make bars that indicate which regions of your gene have been sequenced and place asterisks if you have any clear mismatches. Indicate the position of your mismatch relative to your ATG start codon.

c. If you have a verified mismatch, check to see whether it causes a change in an amino acid of your protein sequence.

d. This information should be incorporated into the next draft of your research paper.

Turn in a copy of your analysis by the end of class Thursday, April 10, 2008.

* +326C to GH to N

1 kbp 2 kbp 3 kbp


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are needed to see this picture.

AttR1

AttR

YFP

MTT1 Promoter


insertion site

Attl1

AttR2Attl1AttR2

Biology 3492 Spring 2008Appendix 3: Assignments, problems sets, and grading sheetsWriting-up your research results (draft 1 due March 6, 2008 -75 pts)The study of epiplasm proteins in Tetrahymena.

As you study the epiplasm this semester, you will be writing a research paper/lab report describing your approaches, methods, and findings from the cloning and analysis of your Tetrahymena gene-YFP fusions, your rtPCR expression analysis, western blot analysis, etc.. Your report should include description of all the steps that you have performed and the results you have obtained from your studies of the protein.

Your report will have the following sections:Introduction: In the introduction, you should include a brief description of the cytoskeleton and a presentation of what is known about the epiplasm in Tetrahymena biology and its relationship to the eukaryotic cytoskeleton. You should also have some introductory material about what the goal of your study is and information that makes the studying the epiplasm feasible and useful to understand cytoskeletal function. It should also contain some description of the genes known to be involved in this process with additional detail about your protein (or class of proteins) if previously discussed in publication. All the relevant background necessary for one to understand your results and discussion sections should be included.

Methods: Here describe what you did. For example, describe How did you clone your gene and then construct your YFP fusions? How did you verify that you had the plasmids that you want? How did you get these fusion constructs into Tetrahymena? How did you visualize the localization? How was the rtPCR expression analysis performed? How did we try to knockdown expression of our genes, and what did phenotypes did we examine? ........ This should include the bioinformatic analysis. This all should be very concise, but have enough information that your lab partners could repeat your experiments. This must be in paragraph form, not as an outline as I passed out. MAKE SURE IT IS IN YOUR OWN WORDS.

Results and Discussion: This is where you present the summary of your results. You should have a more thorough description of your gene’s relationship to genes in other organisms. Include a diagram of your gene with introns marked, indicate your primers used in cloning and expression analysis. Show a diagram of the protein encoded with identifiable domains. If you have a clear ortholog, include a clustalW multi-sequence alignment and describe what you think it tells you (whether or not you have a clear ortholog). Show photos of gels that verify your constructs or tables of the expected and observed sizes of your enzyme digested DNA. Describe them, what were the predictions and how the results fit or do not fit those predictions. Discuss any irregularities in your data. Describe the results from transformation and the eventual GFP localization. Show and describe the results of your rtPCR analysis, western blot analysis and RNAi knockdown experiment. Be sure to describe the rationale of the experimental design. This is most important to readability to the naive reader. What conclusions can you draw from each piece of your data? .... from all your data as a whole? What confidence do you have that your gene encodes an epiplasm component in Tetrahymena. Finish with a description of what you think should be done better or done next

Arrange with Prof. Chalker to get images of your localizations or gel pictures from the lab computers if necessary. Each Student must write their own report, but you can and should consult with one another on the interpretation of the data.

Biology 3492 Spring 2008Appendix 3: Assignments, problems sets, and grading sheetsOral Presentation (100 pts) Tuesday, March 18, 2008; Thursday, March 20, 2008: Prepare a 10 minute presentation using powerpoint.You should describe your approach and results, much as written in your paper. Start with a description of the process that we are studying and how we plan to study this in Tetrahymena. You will want to have an introduction including a description of the relationship between your protein and its homologues of known function. Describe the construction of your fusion plasmid and your GFP localization data. Include results from your western blot analysis and relate that experiment to your GFP localization data. You should try to tell the class what you think it all means.

Dear Class, Just a bit more direction in writing your lab reports. You should think about what are the

goals of each activity that we have performed. In my mind, one could really divide our activities into three distinct parts:

1. The first is characterization of our candidate genes/proteins. This approach was one of bioinformatics. We genes were found by proteomic analysis by Dr. Honts and we are working to confirm his findings using localization data and learn more details about the proteins expresssion and localization.

2. The second experiment is to examine the localization of the protein encoded by our genes by fusing them to a fluorescent protein coding sequence. This will have taken most of our time, and included primer design, PCR amplification to clone the coding region of our genes, followed by LR recombination reactions to complete creation of a plasmid containing our gene-YFP fusion. These will then be electroporated into Tetrahymena and we are now visualizing the localization the next two lab periods. This will also include our Western blot analysis to see that we have full length protein expressed.

3. The third experiment is aimed at determining the mRNA expression level of our genes. This was done by rtPCR and involved the steps of RNA isolation, RNA to cDNA conversion, followed by PCR to visualized gene expression levels. This is as we do not know much about how the cytoskeleton might change during development of Tetrahymena. A positive rtPCR results shows that the gene is expressed and when in the life cycle it is expressed.

You can write your report in either the first or third person. Below are a list of the maim figures/data you should have in your report.

The types of figures you should have for each part (not necessarily exclusive to one part or another):1. Diagram of geneDisplay sequence of gene with introns indicated and sequence of gene cloning and rtPCR primers highlighted.Conserved or Notable domains highlighted


2. Generation of Fluorescent protein fusion: Data to presentPCR amplification of your geneGeneration of your pENTR clones Generation of your pICY clones

Show maps of your desired plasmids constructed (Graphical display)Show gels that confirm you clones (scan in your pictures) and/or represent the data as observed sizes for each lane.Show localization patterns from microscopy of Tetrahymena transformants

If possible show the same cell imaged with white light, YFP fluorescence, and DAPI-stained fluorescence to indicate nuclei.

3. Western blot data of YFP fusion protein expression.

4. rtPCR gene expression gelLabel size markers, tubulin bands (372 bp) and the observed PCR products corresponding

to expression of your gene.

This serves only as a guide, not an outline for your lab report. There is no one correct way to write the report, but we will be looking for whether you understand what each step of our work is intended to learn.

Draft 1 due March 6, 2008 – (75 pts)1. Include an outline of your introduction2. A concise description of the methodologies used to clone and YFP tag your gene and

examination of protein localization.3. Methods of your bioinformatic analyses4. Figure and legends showing: a diagram of your gene, cloning methodology and

electrophoresis data confirming your cloning; Tetrahymena localization images.5. Result sections describing the data in for figure. Written in paragraph form with

appropriate context to help your reader understand what you are doing and why.

Biology 3492 Spring 2008Appendix 3: Assignments, problems sets, and grading sheetsGrading scale for final lab write-up

1. Introduction sets up rest of paperNo purpose described or purpose there but unclear or clearly stated purposepoor context/background incomplete context/background and excellent context1 2 3 4 5

1. Description of methods easy to followLack of several Some methods well described all important elementsmajor elements others not well stated4 8 12 16 20

2. Results described well difficult to understand findings Easy to understand findings4 8 12 16 20

3. Discussion of results No attempt to discuss interesting findings excellent discussion of resultsor indicate problems with experiments and discussion of expectations7 15 24 32 40

4. Graphs, figures, and tables present results wellpoorly labeled/ well labeled/ difficult to decipher appropriate perameters plotted4 8 12 16 20

Clarity of writing:5. Writing indicates clear understanding (good paraphrasing)

Jargon/word/terminology misuse comprehension clearstilted/copied text own words used/clear explanations1 2 3 4 5

6. Sentence structure/Paragraph structure Awkward, poorly stated or easy to read/understandWordy or redundant sentences good transitions1 2 3 4 5

Comments:

Biology 3492 Spring 2008Appendix 3: Assignments, problems sets, and grading sheetsGrading scale for final lab write-up

1. Introduction sets up rest of paperNo purpose described or purpose there but unclear or clearly stated purposepoor context/background incomplete context/background and excellent context5 10 15 20 25

7. Description of methods easy to followLack of several Some methods well described all important elementsmajor elements others not well stated6 12 18 24 30

8. Results described well difficult to understand findings Easy to understand findings6 12 18 24 30

9. Discussion of results No attempt to discuss interesting findings excellent discussion of resultsor indicate problems with experiments and discussion of expectations5 10 15 20 25

10. Graphs, figures, and tables present results wellpoorly labeled/ well labeled/ difficult to decipher appropriate perameters plotted4 8 12 16 20

Clarity of writing:11. Writing indicates clear understanding (good paraphrasing)

Jargon/word/terminology misuse comprehension clearstilted/copied text own words used/clear explanations2 4 6 8 10

12. Sentence structure/Paragraph structure Awkward, poorly stated or easy to read/understandWordy or redundant sentences good transitions2 4 6 8 10

Comments:

Biology 3492 Spring 2008Appendix 3: Assignments, problems sets, and grading sheetsStudent: Grader:Grading scale for presentations

13. Introduction No background Some background given Study introduced fully in biological context 1 2 3 4 5Comments

14. Each experiment introduced with the context of the biological questionLittle rational presented Experimental logic well stated1 2 3 4 5Comments

15. Results described well Figures poorly describeddescription hard to follow Data very well describe1 2 3 4 5Comments

16. Conclusions sound and well described Disorganized presentation of or Easy to understand findingsNo attempt to discuss conclusions excellent discussion of results

1 2 3 4 5Comments

17. Overall Presentation style: Lack of focus in discussion Clear discussion but Very clear presentationDifficult to follow logic some inaccuracy/misunderstanding logical, accurate language 1 2 3 4 5Comments

Biology 3492 Spring 2008Appendix 3: Assignments, problems sets, and grading sheetsGrading scale for lab notebooks -- Midterm

1. A clearly stated purpose.No purpose given purpose there but unclear clearly stated purpose2 4 6 8 10

2. A comprehensible procedure, such that the grader could walk into lab and repeat your experiment exactly the way you did it.3 5 8 12 15

3. A complete record of the results obtained.3 5 8 12 15

4. Thoughtful conclusions.3 5 8 12 15

5. Legibility: 2 4 6 8 10

6. Table of contents/page numbers 2 4 6 8 10

Deductions/comments:

biology 3492: - tgd | tetrahymena genome database …ciliate.org/files/chalker protocols/molecular...

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