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weak bands for methylated DNA - (Sep/09/2009 )

Hello,

I am new to here, so hope that i doing it right. Can someone help me?

I have started with MSP, i am doing it on rat DNA. Using Qiagen kit for bisulfite conversion. Then i am doing MSP PCR, using primers as already published, and very often for MLH1 gene i am getting for methylated DNA weak band (unmethylated is also present and strong and nice), not so strong as in positive control, but for some samples there always is, and negative control and no teplate control are empty and clean?
What it could be? What shoul I do?

Thank you very much

Veronika

-verculka-

how many cycles are you performing? try reducing them.

Nick

-methylnick-

verculka on Sep 9 2009, 11:22 AM said:

Hello,

I am new to here, so hope that i doing it right. Can someone help me?

I have started with MSP, i am doing it on rat DNA. Using Qiagen kit for bisulfite conversion. Then i am doing MSP PCR, using primers as already published, and very often for MLH1 gene i am getting for methylated DNA weak band (unmethylated is also present and strong and nice), not so strong as in positive control, but for some samples there always is, and negative control and no teplate control are empty and clean?
What it could be? What shoul I do?

Thank you very much

Veronika


Sounds like a problem with your methylated DNA. Is there a distributor for methylated rat-DNA or do you use a M.SssI kit to prepare your own methylated DNA?

-MoB-

i am using 65 cycles, is too much?

and, i am using this M.Sssi kit to prepare my own methylated DNA as control

-verculka-

verculka on Sep 9 2009, 11:59 AM said:

i am using 65 cycles, is too much?

and, i am using this M.Sssi kit to prepare my own methylated DNA as control


In my opinion 65 cycles is too much. 35 - 45 cycles should be enough...

Regarding the M.SssI kit: Zymo or NEB? The Zymo kit was pretty well, while I had sometimes troubles with the NEB kit. If you use NEB, increase the concentration of SAM and of MSssI (4 units per g DNA), prepare your own NEBuffer2 without MgCl2 and autoclave the buffer before usage.

-MoB-