LAB 2 Flashcards
steps of PCR
need to use a heat resistant polymerase
Mg2+ is a cofactor for polymerases
PCR buffer: usually at 10X concentration
ensures appropriate ionic strength, pH and osmolarity
factors to consider when designing a primer
20-25nt in length
50% GC content (if higher=mismatching if lower=breakage)
Tm=45-70°C
what is Tm?
temperature at which primers anneal specifically to their complementary DNA -> defined by primer sequence
annealing temperature equation
annealing temp = theoretical Tm=[# of A+T]2°C+[# of G+C]4°C
find the concentration of a given primer equation
site directed mutagenesis theory
lab 2 week 1 protocol
amplify fragments of Nup205 using different combinations of primers
amplify gene fragments of four unknown samples using AMEL primers to determine sex
site directed mutagenesis
what do the master mixes contain
polymerase
forward and reverse primers
dNTPS
H2O with MgCl2
DNA of interest
lab 2 week 2 protocol
mutagenesis efficiency (%) equation
number of blue colonies/total number of colonies * 100
how to determine the fold of amplification with a known efficiency
in theory it’s 2^n where n=number of cycles
but the efficiency is more around 85%
so the calculation should be 1.85^n
drop in efficiency due to the plateau effect
what causes the plateau effect
attenuation of exponential growth rate
1. utilisation of dNTPs and primers (major)
2.stability of reactants (major)
3. competition for reactants by non specific product
4. reannealing of specific product
5. incomplete denaturation or strand separation at high product concentrations
