PCR - L1 Flashcards
Why do we use PCR?
sensitive, cheap, specific, rapid and robust
What are the elements present in a PCR tube
Template, 2 primers, polymerase, dNTP, Mg, buffer
Describe the characteristics of primers
small ss DNA, 6-30 bases, chemically synthesised
State the role of Mg in PCR
Cofactor for enzymes, enhances enzymic activity enabling catalysis
What is the ideal length for primers
18-24
What are primers mainly composed of
40-60% GC
What do primers start and end with
1-2 GC pairs
What is the melting temp
50-60
What is the range of temp that primers should have between each other
5
What end must be complimentary to the DNA
3’
Should primer pairs have complimentary regions
NO
In what form is Mg used
MgCl2
What is the optimal buffer pH
8-9.5
What are the components of buffer
Tris HCL and KCl (or ammonium sulphate)
What are the three stages of PCr
denaturation, annealing, elongation
How many cycles in PCR
30
is a different primer used in the second cycle?
yes
What temp does denaturing occur
95
WHat temp does anneal occue
55
What temp does elongation occur
72
هلا
اهلين
What is the end product of PCR (how much DNA)
1 billion copies
What is the PCR marker and what colour is it
Nancy520 (red)
What type of gel do we use to detect PCR products
agarose gel
