Practicals Flashcards
Recombinant plasmid:
what happens when lac z is functional?
form non-recombinant blue colonies
Recombinant plasmid:
what happens when lac z is disrupted?
form recombinant white colonies
Recombinant plasmid:
What does Ap^r mean?
ampicillin resistance
PCR:
what is the role of Nancy520
intercalating dyes that fit between bases in the DNA helix
Recombinant plasmid:
Why does pblt101 have more bands than plC19H?
recombinant (pblt101) has more bands because it contains the plasmid and the insert
PCR:
if the IP+FUP pair produced PCR what does this indicate?
backwards insert
Supplementary practical:
What is the role of IPTG?
binds to lac repressor and releases it from lac operator = lac operator can now function
SDS page:
What is the role of Sodium dodecyl sulphate (SDS)?
an anionic detergent that disrupts nearly all non-covalent interactions in proteins
SDS page:
What is the role of Tween20?
mild detergent to remove any unwanted proteins or antibodies
Western blot:
what is the function of Tris?
acts as a buffer to maintain the pH
Western blot:
What is the function of sodium in the TBST buffer?
helps reduce non-specific binding on the blotting membrane
PCR:
What is the difference between how a supercoiled plasmid, linear plasmid and circular plasmid look on the gel?
supercoiled= look smaller than their molecular weight bcs they can pass through easily
linear = relative to MW can pass through gel easily
circular = look bigger than MW = take longer to pass through gel
PCR:
Taq DNA polymerase adds nucleotides to which end?
3’ end
Recombination:
what are the key features of the plasmid vector pUC18?
Ampicillin resistance gene,
had a cloning site,
lacZ was there
(disrupted if cloning had happened) and also there was an origin of replication.
