Restriction Enzymes Flashcards
To blunt 5’ overhang
S1 removes overhang
Klenow -DNA Polymerase
Palindrome
Identical on sense and antisense strands when read from 5’ to 3’
To blunt 3’ overhang
S1
T4 DNA polymerase
Isoschizomers
RE from different strains that recognizes same sequence.
What causes no digestion?
Inhibitors present - phenol, salt, EDTA
Enzyme inactive - check exp date
Stability of RE
DNA methyl transferases
Unexpected or extra bands seen after digestion
DNA preparation faulty
Buffer/ enzyme/ water is contaminated
Star activity (RE digests at non-specific sites) high enzyme:DNA ratio.
Why no long digests in water bath
Evaporation changes volume. Do in incubator or PCR machine ( lid heated prevents evaporation)
Applications of REs
Generate recombinant DNA molecules - most NB
Distinguish gene alleles (single nucleotide polymorphisms)
Reporter assays
Restriction fragment length polymorphism
Optimum conditions for RE digestion
pH
Ionic concentrations
Cations (sodium/potassium)
Temperature
RE expensive & temp sensitive, therefore…
Work quickly, on ice.
Return to freezer quickly
Add to rxn last
Make cocktail for many samples
Critical parameters of RE
Purity of DNA (contaminants - RNA, protein, salt, solvents)
Degree of DNA methylation
Optimal conditions of enzyme
Enzyme:DNA ratio
How to account for/ overcome DNA impurity
Increase:
Enzyme
Rxn volume (to dilute contaminants)
Incubation time
Conditions promoting star activity
High glycerol concentration High enzyme/DNA ratio High pH Low ionic strength Organic solvents Substitution of Mg
Options to digest with multiple enzymes
- Both enzymes in both buffers
- Cut with one enzyme, alter buffer cut with second enzyme
- Cut with one enzyme, recover DNA by precipitation, run next digest
How to remove restriction enzyme from reaction
Stop buffer - glycerol, EDTA, bromophenol blue
Heat inactivation
Phenol/ chloroform
Separate with agarose gel, extract DNA
