A little more help with PCR! - (Oct/09/2008 )
This is all the info I have been given on designing a PCR protocol:
PCR reactions typically contain; Polymerase buffer (1x - or 1 fold - concentration), Oligonucleotide 'A' – 0.8 picomolar, Oligonucleotide 'B' – 0.8picomolar, Nucleotides ( a mixture of dATP, dGTP, dCTP, dTTP at 200 micromolar each nucleotide), DNA template (50ng), GoTAQ Polymerase (1.25 units of activity), MgCl2 (2.5mM).
The reaction volume will be 25 microlitres, and you can run a maximum of 5 reactions.
You will be supplied with stock solutions / reagents; 5x Polymerase buffer (5 fold strength), Oligonucleotide A (20pM ), Oligonucleotide B (20pM), MgCl2 (25mM), Nucleotide mixture (10mM each nucleotide), GoTAQ polymerase (5 units of activity per microlitre – use 0.5microlitres per reaction), DNA template (100ng per microlitre) and distilled water.
Question 1: How do I know what volumes I should add of each component?
Question 2: How do I work these out?
Hope this makes some sense! Thanks in advance.
there are semi standard formulation which would be used for a PCR reaction. These reaction mixes are usually defined by the company that makes your polymerase.
The simplest thing to do would to Google GoTaq and find out what is its manufacturer's recommended formulation. Each PCR reaction will have to be optimised by trial and error.
But to give you some idea, here is my standard Taq PCR mix.
5.0 ul 2mM dNTP
2.5 ul 10mM PrimerF
2.5 ul 10mM PrimerR
5.0 ul Taq Buffer 10X
1.0 ul Template DNA (100ng)
2.0 ul 50mM MgCl2
0.5 ul Taq (Activity unknow - made in house)
31.5 ul d Water
50ul - Total
PCR reaction can be as small as 10ul. (You don't need more if it is just for diagnostic purposes)
c1v1=c2v2 for each component to figure out how much of each.
add them all up.
add water to 25ul.
by the way, you will have 2xgotaq in the reaction if you use 0.5ul.