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
0
4 replies to this topic
#2
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Posted 09 September 2009 - 01:26 AM
how many cycles are you performing? try reducing them.
Nick
Nick
#3
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Posted 09 September 2009 - 01:30 AM
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
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?
#4
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Posted 09 September 2009 - 01:59 AM
i am using 65 cycles, is too much?
and, i am using this M.Sssi kit to prepare my own methylated DNA as control
and, i am using this M.Sssi kit to prepare my own methylated DNA as control
#5
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Posted 09 September 2009 - 02:08 AM
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
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 µg DNA), prepare your own NEBuffer2 without MgCl2 and autoclave the buffer before usage.
