Experiemnt 6: Intro Methods for Isolation, Quantification and Analysis of DNA

Plasmids

Small, non genomic, dsDNA, replicons, carry genetic information (usually beneficial)

Replicons

Genetic elements that can self replicate, independent of genome

Purification of plasmid

Mini or maxi prep (depending on scale)

Mini or maxi prep

Series of procedures to purify plasmid

Steps of mini or maxi prep

Resuspension, Lysis, Neutralization, Binding, Washing, Elution

Resuspension

Cells harvest from growth media, Resuspensed in TRIS/EDTA/Glucose/RNAase

Resuspension mixture and purpose

TRIS - buffer and maintain pH, EDTA - prevents DNAase, Glucose - osmotic strength, RNAase - degrades RNA

Lysis

Cell rupture, buffer with NaOH and SDS, degrade and aggregates proteins, denature DNA in single strands

Neutralization

Separate chromosomal DNA from plasmid DNA, small plasmid anneal properly but large chromosomal don’t, chromosomal DNA insoluble (centrifugation)

Binding

Plasmid DNA applied silica-based spin column, promotes salt bridge between sugar phosphate backbone and silica membrane

Washing

Ethanol rich solution, rinses away contaminant and excess salt

Elution

DNA removed from column, low [salt]

molecular cloning

recombining different pieces of DNA, large number of copies

restriction enzyme

cut DNA at specific recognition sequence

DNA digestion

incubation and cutting of DNA by restriction enzyme

Electrophoresis

separate DNA based upon size

migration velocity

speed at which molecules move towards opposite charged poles

migration velocity equation

v=Eq/f, v =velcoity, E= strength electric field, q = net charge on particle, f= frictional coeffiecient

agarose gel

porous solvent filled gel, sieving matrix with minimal interaction with molecules, linear polysaccharidemesh network formed by solidification, smaller moves farther than bigger

calculate mass of agarose in grams

volume of desire (mL) 0.01 * agarose %

DNA ladders

calibrate gel, DNA sample with various pieces of known sizes

gel running conditions

6-10V/cm, high voltage could cause overheating and artefacts (smearing)

Imaging gel

fluorescent based DNA dyes

examples of DNA dyes

RedSafe, SYBER safe, HydraGreen, SYBR Gold

Analysing DNA quality

average pieces >10kbp, size of smear (eukaroytic will always smear)

analysing DNA purity

absorabance ratio of 260/280

Confirming PCR success/ DNA isolation

check if DNA size is as expected

PCR

make many millions of copies of DNApiece