Basic question about setting up a PCR reaction - (Oct/09/2008 )
Hi I am an undergrduate and we have been asked to carry out a PCR reaction. Here is the info we were given:
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.
Now we were asked to find out the optimum Mg concentration for the reaction. So is it best to add say 5 different final concentrations of MgCl2 to a supermix of the other chemicals and see which gives the best result on a agarose gel?
Sorry I am not very good at PCR! Any help would be great.
This is exactly what to do. Just make sure that the total final volume of the reactions are the same even when you add different amounts of MgCl2, and that the final concentrations of buffer, oligos, etc. are correct.
Glad to be of help.
As a general rule, whenever you are setting up an experiment to optimise something, you make sure that there is only ONE variable that changes. This is true in setting up PCR, or doing cell culture studies, or just about any other type of experiment.
For PCR or other enzymatic reactions, this often means each tube with will need a different amount of water to ensure the final volume will be the same between samples. I know this sounds like a pretty basic piece of advice, but just two days ago a senior PhD student in my lab was setting up a set of reactions and forgot to re-calculate the different amount of water she'd need to add to each sample to get the correct final concentrations. Never forget to do this!
Thanks I will remember that for sure! It has been a bit difficult as we are only undergrads and its the first time most people have had to plan a PCR protocol from scratch with only a list of chemicals to go on.