SDM Flashcards
0
Q
Primers in mutagenesis can
A
Substitute
Delete
Terminal substitution
1
Q
Aspects that can be studied by SDM
A
Enzyme mechanism Aspects of protein structure (does glycosylation affect fx) Protein engineering (GFP dif colours) Alter substrate specificity (drugs)
2
Q
Two mutagenesis approaches
A
Quick change
SOE PCR
3
Q
Describe Dpn1 digestion
A
DAM methylase adds methyl groups onto GATC. PCR amplifies unmethylated DNA. Dpn1 digests methylated DNA therefore non mutated DNA is eliminated
4
Q
Adv of QuickChange
A
Fast
No intermediate DNA purification needed
Only amplifies parent DNA
5
Q
Disadv of QhickChange
A
Whole plasmid amplified Long primers needed Needs Dpn1 / methylation dependent Can lead to insertion of multiple primer sequences Relatively inflexible
6
Q
Adv of SOE PCR
A
Short amplicon size
Allows complex mutagenesis / flexible
No Dpn1 needed
7
Q
Disadv of SOE PCR
A
Labour intensive/ slower
Multiple PCRs = more likely to have unwanted mutations
Need unique restriction site for final plasmid
8
Q
NB checks after SDM
A
Restriction enzyme digest
DNA sequencing
