DNA extraction, electrophoresis and RE digests Flashcards
DNA extraction overview
Sample > add PBS > sucrose triton X-100 > TE buffer > SDS > proteinase K > incubate overnight > NaCl > alcohols > air dry
Three common methods for extracting DNA
Phenol-chloroform extraction
Silica based kits
Proteinase K and salting out
What protects bacterial DNA from restriction endonucleases
Methylation
Define isoschizomers
A pair of REs that recognize the same site and cleave at the same position
Define neoschizomers
REs that recognize same sight but cleave at different posititions
How do you ensure REs don’t get damaged
Work quickly
Work on ice
Add to mixture last
Factors affecting RE digests
Purity of DNA (protein, salt, RNA, solvents)
DNA methylation
Reaction conditions (pH, temp, ionic strength, concentration)
Enzyme to DNA ratio
How to improve DNA purity in RE digest
Purify DNA
Increase amount of RE
Increase reaction volume (dilute contaminants)
Increase incubation time
Possible causes of star activity
High glycerol (>5%) High enzyme:DNA ratio (>100units/ug) Low ionic strength High pH (>8) Presence of organic solvents Substitution of Mg with other divalent cations
Separation in gel electrophoresis based on
Size
Charge
Conformation
Types of gel electrophoresis
Agarose - DNA Formaldehyde - RNA PAGE - DNA SDSPAGE - protein Pulsed field electrophoresis - large DNA Capillary electrophoresis
What is agarose
Linear polysaccharide
Seaweed extract
Basic repeat unit = agarobiose
Role of electrophoresis buffer
Maintains pH
Provides ions for conductivity
(Tris acetate/tris borate)
Three purposes of loading buffer
Increase sample density (glycerol/sucrose/Ficoll)
Adds colour to sample
Contains dye to monitor progress of gel (bromophenol blue [linear ds 300bp] xylene cyanol [linear ds 4kb])
What is ethidium bromide
Planar molecule that intercalates between nucleic acid bases.
Fluoresces under UV light
Factors influencing DNA migration on gel
Molecular size of DNA
Agarose concentration
DNA conformation
Applied voltage
Dense agarose gel separates …
Low conc agarose gel separates …
Smaller DNA fragments
Larger DNA fragments
Mobility of super coiled DNA relative to linear depends on..
Agarose concentration
Strength of current
Ionic strength of buffer
At high voltage ..
Large fragments run proportionally faster than smaller ones. (No more than 5V/cm)
What is different about RNA gel
RNA tends to form 2dry structures Contain denaturants (formaldehyde, formamide) All reagents/ apparatus treated with DEPC (diethyl pyrocarbonate) to remove all RNAses
Difference between agarose and PAGE
Page has greater resolution but smaller range of fragments
PAGE (5-500bp) differentiate >1bp
Agarose (0.1-25kb) differentiate >20bp
Define star activity
Relaxation or alteration of restriction enzyme mediated cleavage of DNA. Results in cleavage at non-canonical recognition sites.
Difference between TAE and TBE
TAE - higher electric current = more heat = poor gel banding pattern. Good for short gel run times. LOWER buffering capacity
TBE - high resolution of DNA fragments, not recommended if wanting to extract/purify DNA. HIGH buffering capacity.
How is PAGE gel formed
Acrylamide monomer forms chains in presence of free radicals
Chains become cross linked
Process stabilized by TEMED and inhibited by oxygen.
NOTE acrylamide is a neurotoxin
What are non-denaturing PAGE gels used for
Separating and purifying dsDNA fragments.
Detecting small nucleotide differences.
What are denaturing PAGE gels used for
Separating and purifying ssDNA frags
Agent (eg urea or formamide) prevents base pairing
Downstream - sequence analysis, genotyping, isolate radiolabelled probes.
How to visualize DNA in PAGE
Radioactivity (32P, dried under heat and vacuum, x-ray film) Silver staining (silver solution binds DNA, reacts with formaldehyde in alkali - looks brown on yellow)
