CTE Skills Test Review

LAB ATTIRE

Closed toe shoes Lab coat Goggles Gloves Hair tied back No cosmetics No gum, food, or drink

DISPOSAL

Glass Glass waste disposal box

Bacteria Bleach Autoclave

Other waste Proper garbage disposal

LAB NOTEBOOK

Date Initials Number each page Table of contents

LABELING

Reagent bottles, bacterial plates, etc. Initials Date What is in and/or on Concentration (M or %) On the bottom of the plate [part with agar]

ASEPTIC TECHNIQUE

Disinfect counters and work surfaces. Flaming loop, spreader, etc.

DISINFECTION

Always clean counter before and after Use

70% ethanol Bleach Other disinfectants

EFFECT OF UV ON BACTERIA Kills bacteria if you leave it under UV light

AUTOCLAVE

Uses high heat and pressure to sterilize objects

MICROPIPETTES

1000 ul = 1 ml Be within the range but not on the end with

amounts to pipette P20, p100, p200, p1000

CHEMISTRY EQUATIONS Molarity (M) = moles/liter moles = grams/molar mass

Molar mass is the sum of the atomic mass of elements

M = [grams/molar mass]/liter Volume % = volume solute/volume solution x

100 M1V1 = M2V2 C1V1 = C2V2

BONDS Hydrogen- hydrogen bond with other element

Water, DNA between nitrogen bases, proteins Covalent- sharing of a pair of electrons by two

atoms DNA on backbone

Ionic- through transfer of 1+ electrons ions Buffers- salt, etc.

Disulfide- binds peptide chains Gives protein 3D shapes [usually between chains]

Peptide- bond amino acids, remove a molecule of water (dehydration) Amino acid bonds

HYDROPHOPIC/HYDROPHILIC Part of the membrane Hydrophobic

Water fearing Tails

Hydrophilic Water loving Head

POLAR/NON-POLAR

Polar Has a charge

+/- Ex: water

Non-polar Doesn’t have a charge

Ex: sugars, oils

pH

Salt concentration can change the shape of proteins

Change in acid and bases can kill enzymes Acids

Lower pH to 1>7 Bases

Raise pH to 7<14

STOCK SOLUTIONS

Dilute stock solution to get desired solution concentration

C1V1 = C2V2

SPECTROPHOTOMETRY

An instrument used to determine the intensity of various wavelengths in a spectrum of light

Can determine concentrations of solutions Can make a graph of OD and absorbance v.

concentration Can change the wavelength to find the protein,

DNA, RNA, and bacterial concentration

SERIAL DILUTION

Dilute to get smaller and smaller concentrations Can go from lawns to single colonies, an alternative

to streaking

PROKARYOTIC V. EUKARYOTIC Prokaryotic cells

No nucleus Eubacteria Archaebacteria- extreme bacteria Operon (grouped genes) Don’t have introns

Eukaryotic cells Nucleus Protists Fungi Plants Animals DNA has introns and exons (splice introns out and bind

exons together to form mRNA)

MEDIA PREPARATION & INCUBATION Media preparation- can manipulate what you grow Incubation

37°C if grown inside body 25°C if grown elsewhere

LB/AMP/ARA LB- nutrients necessary for bacterial growth AMP- ampicillin resistance to see if there was an uptake of

the protein Selective procedure- only ampicillin resistant bacteria can

grow ARA- arabinose to turn on the GFP

Allows the gene to be transcribed (operon involved)

ANTIBIOTIC RESISTANCE FOR SELECTION Antibiotic- natural substance secreted by 1

microorganism that will kill or inhibit growth and reproduction in other microorganisms

Shows if there was an uptake of the new genes

INOCULATION OF MEDIA

Streaking Proper lab attire…not that kind of streaking! Get a single colony on you loop and streak it

across the plate in a zig-zag fashion. Turn it a quarter turn, flame and repeat.

Spreading Pipette LB broth onto plate and sterilize paperclip.

Spread broth with paperclip over the entire gel.

IDENTIFYING MICROORGANISMS Colony Morphology

Size Shape Margin Elevation Texture Light transmission Color

Antibiotic resistance Incubation temperature Gram staining

BACTERIA TYPES

Cocci round

Bacilli rod

Spiral Staph-

Grape-like clusters Strep-

Chains Diplo-

Two together

GRAM STAINING

Depends on the structure of the cell wall

Gram positive Purple

Gram negative Red

DNA STRUCTURE

Runs 5’ to 3’ Sugar (deoxyribose) Phosphate (phosphoric acid)

Negative charge (allows for electrophoresis) Nitrogen bases

2 hydrogen bonds Adenine – Thymine Gene cutting happens most often here

3 hydrogen bonds Guanine – Cytosine

DNA REPLICATION ENZYMES Helicase

Splits the DNA molecules apart RNA primase

binds primers (RNA nucleotides) by complimentary base pairing

DNA polymerase Adds nucleotides to extend the DNA Binds leading DNA strand starting at 3’ end

TAQ polymerase can withstand high heat RNA primers are removed Ligase

Seals gaps in the sugar phosphate backbone

RNA

Ribose sugar has one more oxygen molecule

Phosphate Nitrogen bases

Adenine – URACIL not thymine

Guanine – Cytosine

TRANSCRIPTION

DNA to RNA Eukaryotes

Eukaryotes have introns and exons; the introns are removed

5’ cap and 3’ poly-A tail on the exons that have been spliced together

Prokaryotes Operons

PROTEINS

Peptide bonds Eukaryotic

Multiple proteins from 1 RNA Prokaryotic

Operon

PROTEIN STRUCTURE

Primary Amino acid sequences

Secondary Alpha-helices or beta-sheets

Tertiary Domains

Quaternary Subunits

FUNCTIONS OF PROTEINS- REST! Regulatory

Genes and cellular processes are turned on and off Enzymes

All enzymes are proteins but not all proteins are enzymes Covalent bond breakage and formation

Structure Mechanical support to cells and tissues

Transport Move things in and out of the cell

PROTEIN SYNTHESIS RNA to protein Initiation Elongation Termination

The stop codon doesn’t code for an amino acid

mRNA coded DNA Codons

tRNA transfers amino acid Anti-codons

Amino acids

AMINO ACID CHARACTERISTICS Peptide bonds (dehydration) Water

Hydrophilic Hydrophobic

Charge Positive Negative

Polarity Polar Non-polar Uncharged polar

DNA FINGERPRINTING

Identifies matching DNA fragments (bands) RLFP- Restriction length

fragment polymorphisms VNTR- Variable

Nucleotide tandem repeats

Introns- non-coding sequence that varies

RESTRICTION ENZYMES

Also called endonucleases Specific sequences of DNA nucleotides Cut at specific places Can be palindromes (same forwards and

backwards) Come from bacteria

To cut up viral DNA Methylate own DNA to protect it

RESTRICTION DIGEST

Where you cut DNA with restriction enzymes Results in DNA fragments Can then be run on gel electrophoresis

GEL ELECTROPHORESIS Gel electrophoresis- uses electric current to

separate DNA fragments by size Runs to red (positive) Bands

Various sized fragments of DNA Introns

Pieces Smallest ones run the farthest

SDS-PAGE ANALYSIS

Run vertically Mostly used for protein Smaller pores than agarose

Smaller matrix Sorts by size and charge

PCR

Polymerase Chain Reaction Denature

Raise the temperature to unzip DNA Anneal

attach primers Extend

Binds TAQ polymerase TAQ polymerase can withstand high heat

Heat and cool 1 million copies for 30 cycles

DNA SEQUENCING

Used to know the nucleotide sequence of the human genome

Process Put DNA with DNTPs

Terminates elongation of DNA PCR Run on a gel to tell the sequence of the

nucleotides

READING FRAME

Frame shift mutations Point mutations

Deletion Insertion

RECOMBINANT DNA

2 different pieces of DNA combined Use restriction enzymes to cut the gene of interest

and the plasmid Insert the gene of interest into the plasmid

TRANSFORMATION

Insertion of a gene into bacteria Competent cells- take up the plasmid Restriction enzymes- cut the plasmid Selection- so you can get the colonies with the

selected gene Antibiotic resistance

PLASMID Origin of replication

Allows plasmid to self-replicate Antibiotic resistance

Allows for the selection of the desired bacteria Operon

Turns on the gene of interest Gene of interest

Example: GFP