How to increase the product amount in BSP? - weak amplification in BSP (Dec/15/2008 )
Please help me to work it out!
I am checking the methylation status of fwa locus in Arabidopsis by Bisulfite sequencing, but I can just get very little amount of PCR products. The expected product is ~ 500bp, and the primers are degenerated. I used Qiagen's Epitect kit for bisulfite conversion, used common Taq polymerase, and tried all my known methods, but still the band I got was very faint, and heavy dimer amplification.
I raised dNTP (0.125 mM) and primer (1uM) concerntration, used hot start PCR combining with touch down PCR (72 degC -->54 degC ), more Taq (1ul (2.5U)/50ul), more cycles (40).
Should I try Taq of another company? or just add more DNA template? or even more cycles? any suggestion is appreciated
You can reamplify your PCR product using the same primers. The 2nd round PCR can cycle for ~30 cycles.
where did the primer sequences originate from? are they reliable and do they work?
THX! PCRMAN and Nick!
PCRMAN, Won't the 2nd PCR bring more errors? isn't it better to try nested PCR in this case ?
I have been successfully got enough amount of PCR for cloning for some samples, but for other sample, there is no luck. So how much bias could be generated in 2nd round PCR? Will the difference of 1 round and 2 round result be significant? I afraid inconsistent protocols might be a problem in publishment.
The primer sequence was from published paper, and I had contacted him about the PCR conditions, he did think it is not easy with these primers .