Q regarding primer mix for MSP!! - (Nov/02/2012 )

Hi,

I am a Phd student, my project based on study DNA methylation in small cell no. I have found this paper which stated primers, they used similar sample type. (table in page 534) I want to order the primers, but i have a Q when i run my MSP, which primer i used in first run and which i will use in my 2nd run? I am really confused!

paper title:Characterization of a New NIH-Registered Variant Human Embryonic Stem Cell Line, BG01V: A Tool for Human Embryonic Stem Cell Research
sorry i can't attach the paper! but i copy past the primer that i interested in...





H19 promoter region L-Unmethylated primer L-Methylated primer
5􏰅-TTTTAGGAATGTGAGGTTTGAGTT-3􏰅 5􏰅-TTTAGGAACGTGAGGTTTGAGTC-3􏰅



H19 promoter region R-Unmethylated primer R-Methylated primer
5􏰅-CCTACTACTCCCTACCTACCAACAC-3􏰅 5􏰅-CCTACTACTCCCTACCTACCAACG-3􏰅



I hope find the answer !

regards,

Laila

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.

hi,

Thanks for ur answer, in each run i need to use the original DNA sample?

I can't do nested PCR? with these primer?

laila

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.

hi,

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

3-step cycling
denaturing 15s 94c
Annealing 30s 50-55c
Extension 30s 72c
44cycles

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,

Laila