IGEM 101: Session 3 3/12/15Jarrod Shilts 3/15/15Ophir Ospovat.

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Presentation transcript:

iGEM 101: Session 3 3/12/15Jarrod Shilts 3/15/15Ophir 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% ~ 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 PlasmidLinear PlasmidSupercoiled DNA Single-Stranded Plasmid Much Higher HigherNativeLower

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 V Use guide RNA to target specific, custom sequences (CRISPR)

Nomenclature Derivation of the EcoRI name AbbreviationMeaningDescription EEscherichiagenus cocolispecific epithet RRY13strain IFirst identified order of identification in the bacterium 5‘ GAATTC 3’ 3‘ CTTAAG 5’ 5‘ GAATTC 3’ 3‘ CTTAA G 5’

Restriction Digest Ingredients 1.DNA 2.Enzymes (1 or more) 3.Buffer Solution 4.Water (ddH 2 O) Buffer Composition 1.Potassium Acetate 2.Tris-acetate 3.Magnesium Acetate 4.BSA 5.Beta-mercaptoethanol