Today

• Do you have PCR amplicons?

• Run gel

• Background DNA and our PCRs

• Interpretation of PCR results

• What to do next?

15 minute powerpoint topics(G+) date topic name

21-Sep Discovery of DNA structure Janette Mendoza

25-Sep Restriction enzymes Gabriela Perales28-Sep Southern blotting Carlos Garcia G2-Oct Cloning Timothy McBride G

6-Oct The first sequenced gene Conrad Greaves

13-Oct (q)PCR, specificity and sensitivity

Krystal Charly

16-Oct ESTs Ian Keller

20-Oct BLAST and database searches Ryan Heimroth G

23-Oct Microarrays Bianca Myers26-Oct Forensics Jennifer Gutierrez

30-OctGenome sequencing , the

$1000 genomeAyesha Arefin G

2-Nov Next generation sequencing Leslie Janet Lopez G

6-Nov Bioinformatics Amalia Parra9-Nov Epigenetics Clyde Moya

13-Nov non-coding RNA Helen Nordquist13-Nov C-value paradox Kelsey Cook G

20-Nov Phylogenetic genomics Jennifer Cooksey

23-NovGenes associated with Type 1

diabetesKatie Kesler

http://sev.lternet.edu/about

FIELDTRIP to Sevilleta LTER, Sample collection:

Sunday 13 September

(Sunday 20 September)

PARASITES AND SNAILPARASITES AND SNAIL BIOLOGY BIOLOGY

“identity, possibilities”phylogenetics

“intentions”transcriptomics

PCRrDNA/mito

BioanalyzerDNA-free,

direct sequencing

gel electrophoresisnanodrop spec

Sequence ID (BLAST)editing

Phylogenetics

electrophoresisRT-PCR

gel

CTAB/DNAzol

Trizol

TA cloning, B/W screening

M13 sequencing

Primer design, walking

Qiagen plasmid extraction Restriction digests

DNA

RNA

GenBank submission

Today• Use your notes/handouts • Make 1% agarose gels, 1 per 2 groups

(i.e. 1+2; 3; 5+6; 7+8; 9+10 )• Analyze 10 microliter of each of your 4

PCR reactions. Use layer buffer with GelRed (how much?)!

• Lecture• Results

MW MW

uneven groupPCR reactions

1-4

even groupPCR reactions

1-4

Make 1% agarose gels,1 gel/2 groups, (i.e. 1+2; 3; 5+6; 7+8; 9+10)

Use 12 well combsAnalyze 10 microliter of

each of your 4 PCR reactions.

Use layer buffer with GelRed (how much?)

5l marker/lane

Uracil lacks this group

Difference between hydrogen bonding (weak) of base pairs and covalent bonds (strong) of backbone extremely important

Biologicallyinformation is “stored” AND

can be accessed and transferred

In laboratoryds DNA can be denatured and reannealedwithout losing information

Fig. 6-15

DNA synthesis is 5’ to 3’ andsemiconservative, DNA polymerase has a proof readingcapability.

DNA replication = DNA synthesis (template = DNA)

(transfers information from generation to generation--cell and organism) Primer required

Transcription = RNA synthesis (template = DNA)

(transfers information from DNA to cellular metabolic machinery)

Promoter not primer required

RNARibonucleic acidAUCGsingle stranded (5’-3’) many copies/cellvariable populationbreakdown (hydrolysis)shortmessages, regulators, transporters

DNA Deoxyribonucleic acid ATCGdouble stranded (5’-3’)one copy/cellconstant stablelongprotein encoding, introns, regulatory sequences intergenic junk(?)

NOTESProtocol gel-electrophoresis?

Can you independently repeat the PCR?

Do you know what to expect?

NOTESProtocol gel-electrophoresis?

YES

Can you independently repeat the PCR?

Do you know what to expect?

NO and NO

Because you were not given the needed information for the PCR reaction,the primer sequencesor size estimates of the anticipated amplicons

PCR details

• AmpliTaq Gold,4 mM MgCl2

• 1 mM of each primer (50 picomoles)

• Cycling profile– 10' 95C (hot start)– 30x (30" 95C; 60" Tm; 60"+5" 72C)– 7' 72C,– ∞ 4C

PCR details• Primers:

• 16S and CO1– 16S Tm 55C expected size ~600bp

• 16SAr: 5'- CGC CTG TTT ATC AAA AAC AT -3’• 16SBr: 5'- CCG GTC TGA ACT CAG ATC ACG T -3’

(Palumbi, S. R. 1996. Nucleic acids II: the polymerase chain reaction. In: Molecular Systematics (eds. Hillis, D. M., Moritz, C. and Mable, B. K.), pp. 205–247. Sinauer & Associates Inc., Sunderland, Massachusetts.)

– CO1 Tm 48C expected size ~700bp

• LCO1490: 5'-GGT CAA CAA ATC ATA AAG ATA TTG G -3’• HC02198: 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’

(Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994 DNA primers

for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates. Molecular Marine

Biology and Biotechnology, 3, 294–299)

PCR details• Primers:

• "parasite" rDNA 18S and 28S (Olsen et al. 2003). – 18S Tm 50C expected size ~1800bp

• wormA: 5'- A/GCG AAT GGC TCA TTA AAT CAG -3’• wormB: 5'- ACG GAA ACC TTG TTA CGA CT -3’

– 28S Tm 50C expected size ~1400bp• LSU: 5'- TAG GTC GAC CCG CTG AAY TTA AGC A -3’• 1500R: 5'- GCT ATC CTG AGG GAA ACT TCG -3’

PCR interpretation

Band?Single/multiple?

Strong/weak?Correct size?Positive ID?

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