Q regarding primer mix for MSP!! - (Nov/02/2012 )
You need to run two PCRs in parallel with one using the Unmethylated pair (L+R) and the other using the methylated pair. These primers are not for nested PCRs.
Thanks for ur answer, in each run i need to use the original DNA sample?
I can't do nested PCR? with these primer?
The template DNA should be bisulfite treated, although you can include unmodified DNA as controls.
You can do nested PCR, but you need to design an additional primer pair outside the above primers you give. The outside primers a.k.a. universal primers, will amplify bisulfite modified DNA only without bias toward either methylated or unmethylated DNA.
I am going to order my primers from SIGMA, it's need to be desalt primer?
if i used bisulfited treated DNA and use the above primers to run two parallel PCR with the following cycling protocol (I will use the EpiTect MSP from QIAGEN)
initial activation step: 10 min 95c
denaturing 15s 94c
Annealing 30s 50-55c
Extension 30s 72c
final extension 10 min 72c
My reaction composition will be:
25ul Eptic master mix(contain the hot start Taq, d-tec polymerase,Tris.cl, KCl (NH4)SO4, MgCl2 and dNTPs), I ul from each primer ( methylated and Unmethyalted), 1 ul DNA template (bisulfited) and 23ul RNase-free water
Do u think this make sense? and can work and give visible band?
Thanks for ur great help,