PCR

Question 1

What are the main components needed for PCR?

Answer 1

Taw polymerase
dNTPs
Oligonucleotide primers
MG2+ ions

Question 2

What is the general process?

Answer 2

Denature DNA
Anneal primers
Elongate primers

Question 3

How should primers be designed?

Answer 3

~20 bases long
GC content should be 45=55%
Annealing temperature should be within ~1 degree Celcius of each other
Primers must not base pair with each other or themselves or form hairpins
Must avoid repetitive DNA regions

Question 4

How to optimise the PCR reaction

Answer 4

The annealing temperature of primers 
[Mg2+]
Extension time 
Denaturing time
Annealing time 
Extension temperature 
Amount of template

Question 5

How to optimise annealing temperature?

Answer 5

Primers have a calculated annealing temperature but must be confirmed practically in temperature steps 2 degrees Celcius above and below using a gradient cycler

Question 6

How to optimise [Mg2+]

Answer 6

The fidelity of PCR depends on [Mg2+] so vary in steps of 0.5mM
Sometimes a compromise between yield and specificity

Question 7

What are hairpins?

Answer 7

Sometimes a primer may be self-complementary and is able to fold into a hairpin

Question 8

What are dimers?

Answer 8

A primer may form a dimer with itself or other primers and form unwanted substrates for Taq polymerase