iGEM 101: Session 3

3/12/15 Jarrod Shilts3/15/15 Ophir Ospovat

3a. Gel Electrophoresis

Use to separate macromolecules by size and charge

Different percentages allow for different resolutions and specificity

Ingredients are TAE Buffer, Agarose, and Ethidium Bromide

Ethidium Bromide is highly toxic as well as mutagenic and teratogenic

Mechanics of Electrophoresis

▪ DNA is negative and is attracted to anode (red)– Always RUN to RED!

▪ TAE Buffer provides ions for current– Tris pH 8.0– Acetic Acid– EDTA

DNA Visualization

Fluorophores

▪ Intercalators– Ethidium Bromide– GelRed

▪ SYBR– SYBR Green– SYBR Safe

▪ Methylene Blue

Non-Fluorophores

▪ Crystal Violet

DNA Resolution

Different gel percentages result in different resolutions with the most resolution at higher agarose ratios.

0.7% ~ 5-10 kb

2% ~ 0.2-1 kb

Ladder/Marker

A DNA marker is a combination of DNA segments of known sizes along with several dye fronts that allow tracking the progress of the gel as well as determining the size of bands during imaging.

Plasmid Movement in a Gel

Nicked Open-Circular Plasmid

Relaxed Plasmid Linear Plasmid Supercoiled

DNA

Single-Stranded Plasmid

Much Higher Much Higher Higher Native Lower

3b. Restriction Endonucleases

▪ Enzymes that cut DNA at specific site– 2 cuts through strand by nucleophilic attack

▪ Originally form of innate immune system in prokaryotes

▪ Several different classifications– Type of Cut (sticky vs. blunt)– Type of recognition site (palindromic, inverted)

*Enzymes that recognize the same sequence: neoschizomers

*Enzymes that cut the same sequence: isoschizomers

Types of Restriction Enzymes

Type I

Cuts at a great distance away from recognition site, often random

Type II

Cut within or very near to recognition site, usually dimers

Type III

Cuts short distance away from recognition site, recognizes non-palindromic inverse sequences

Type IV

Target modified DNA such as methylation, hydroxymethylated, etc.

Type VUse guide RNA to target specific, custom sequences (CRISPR)

Nomenclature

Derivation of the EcoRI name

Abbreviation Meaning Description

E Escherichia genus

co coli specific epithet

R RY13 strain

I First identifiedorder of identificationin the bacterium

5‘ GAATTC 3’

3‘ CTTAAG 5’

5‘ G AATTC 3’

3‘ CTTAA G 5’

Restriction Digest

Ingredients

1. DNA

2. Enzymes (1 or more)

3. Buffer Solution

4. Water (ddH2O)

Buffer Composition

1. Potassium Acetate

2. Tris-acetate

3. Magnesium Acetate

4. BSA

5. Beta-mercaptoethanol