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BIO2030 Biotechnology > PCR > Flashcards

Flashcards in PCR Deck (8)
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What are the main components needed for PCR?

Taw polymerase
Oligonucleotide primers
MG2+ ions


What is the general process?

Denature DNA
Anneal primers
Elongate primers


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
Extension time
Denaturing time
Annealing time
Extension temperature
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


What are dimers?

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