Help! No bands after BSP!
Posted 08 August 2012 - 01:41 AM
I've been doing BSP recently. But I got a problem that I cannot get any bands after bisulphite specific PCR.
I used EZ DNA Methylation-Gold™ Kit to treat gDNA. The amount of treated gDNA is around 1.5 ug. I didn't digest gDNA before the treatment. The concentration of gDNA after treatment is around 50~80 ng/ul. And I always use 3ul to run PCR.
The specific primers has been used from a published paper. So I think they might work well.
the gDNA is down here:
the methylated target sequence:
the primers are :
I used hot start Taq to run PCR and the annealing temp is 55.
I also tried a Touchup PCR with the annealing temp changing from 50 to 60, then 55.
but I cannot get any band still. could someone help me?
Posted 11 August 2012 - 11:43 AM
Posted 08 February 2013 - 03:53 AM
You can try to decrease the extension temperature to 65C and increase the time for 2 minutes. It really helped me
Posted 11 February 2014 - 11:43 AM
Hi, I have been having troubles with my MSPCR for a long time . I tried to establish the best temperature for both Methylated and Unmethylated PCR products...
I used a cell line that is already reported that should be positive for both events (SNU423 hepatocellular carcinoma, which has one allele methylated and the other unmethylated).
After the genomic DNA extraction I perform the Bisulfite treatment and used this treated DNA for MSPCR using primers specifics for Meth and Unmeth.
I have bands in the methylated one but I dont obtain any bands in the unmethylated which is not in according to the literature...
What could be happening ??????