strang phenonmenon in MSP experiment - (Dec/16/2008 )
I have done a successed MSP experiment two days ago, but now curiously, when I do the same experiment now, I can not amplify any band with totally same reagent, even on the positive control. Please help me to solve that. I am frustrated with that problem for long time. The following is detail:
I work on the mouse genomic DNA.
My prime of MSP is salt-free purification, but dissolve in the water for 100um stock solution, and diluted to 10um storage solution.
my product is about 100 bp to 200bp, is very short.
My bisulfite treatment is similar with http://www.protocol-online.org/forums/inde...?showtopic=9375, but I use Qiagen PCR purification kit and I do wash after the last step. especially, I use tRNA and sodium acetate but not ammonium acetate suggested by methylnick. So I am not sure the recovery rate of Bisulfite DNA is high.
I have changed the fresh everything, incuding water and primer (but same stock solution), but it does not work.
Additionally, My MSP is just one PCR for 40 cycles, but not nested.
I think about it, and there is several possibilty: one, the primer is degraded in short time, but when I got fresh work solution, it still not work. I am wondering whether the stock solution should be degraded. Are you storing the stock solution in TE (ph8.0)?
two, my bisulfite treated product is less producted, so the it is degraded very soon. I also dissovled in the water, wihich is same with protocol suggested. I don't know whether the DNA precipitation is so less effective.
Please give me diganosis, thank you very much.
Actually, I have got excellent result for the first time, but things became terrible after just 4 times.
what gel should be used for MSP product running? just common agrose gel or TBE-acrymide gel, the later should be high sensitive, but too laborsome.
what enzyme do you use? only just platinum Taq, or the common taq enzyme can be used. the former is too expensive!
thank you very much
Hi, where is methylnick? hope you can help me!