Flashcards in PCR Deck (8)
What are the main components needed for PCR?
What is the general process?
How should primers be designed?
~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
How to optimise the PCR reaction
The annealing temperature of primers
Amount of template
How to optimise annealing temperature?
Primers have a calculated annealing temperature but must be confirmed practically in temperature steps 2 degrees Celcius above and below using a gradient cycler
How to optimise [Mg2+]
The fidelity of PCR depends on [Mg2+] so vary in steps of 0.5mM
Sometimes a compromise between yield and specificity
What are hairpins?
Sometimes a primer may be self-complementary and is able to fold into a hairpin