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BIO2030 Biotechnology > PCR > Flashcards

Flashcards in PCR Deck (8)
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1

What are the main components needed for PCR?

Taw polymerase
dNTPs
Oligonucleotide primers
MG2+ ions

2

What is the general process?

Denature DNA
Anneal primers
Elongate primers

3

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

4

How to optimise the PCR reaction

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

5

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

6

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

7

What are hairpins?

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

8

What are dimers?

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