2 replies to this topic

[YYZDNA]

Hi.
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:
CCAGGCAGTCCCCCAACTGTAAGGAAGACTCGTGTATGTATGTGCATATGTGCATTTCCCCAGGGAAAAACATCCACAGCTTCCATG
ACGAGAGGGGTCGTGACCCCTCCCCGCCAAAAGATTAAGGACCTGCGATCCTACAGACCGGAGCCCTGTTTGAAGTCTGCGTTGC
CCCTCACCTCAAGCTGGTCACTGTGTGAAGTTGGCCTAGAATCCCCCGGCCCCTGGGAGCTTGTTCCTCCGCCTGTAAAATGGGG
CTGCAGGGCCGTCCACGCGGCCACCGGAAGGACAAGGTGTTCAGGCCGCTAGGCCGCTCCCTGGCAAGCGATTCCCACTCGCAG
CGCGGCCTCGACCCTCGCCCAAGACGCGCCCTCCGCGCCCCCACCCCCTCCAGGCCCTGGCCAGTCCACCTCCCGCTTGGGGCG
GCAATTTGTCTCCTTTTGAACCCCCCGCCCCCGACGGGTTTCCCCCTTTGATTCGCGGCCCGGAGGCTTCCCCCCGCTTTGAAATG
CAAACCCGCCTCGGCTGGGGCCGCGGGCGGCCCGGAGCTATAAAAGGCCTGGGTGGGGCGGGCGCGGCGGCAGGACAGCCGAG
TTCAGGTGAGCGGTTGCTCGTCGTCGGGGCGGCCGGCAGCGGCGGCTCCAGGGCCCAGCATGCGCGGGGGACCCCGCGGCCACC

the methylated target sequence:
TTAGGTAGTTTTTTAATTGTAAGGAAGATTCGTGTATGTATGTGTATATGTGTATTTTTTTAGGGAAAAATATTTATAGTTTTTATGACGA
GAGGGGTCGTGATTTTTTTTCGTTAAAAGATTAAGGATTTGCGATTTTATAGATCGGAGTTTTGTTTGAAGTTTGCGTTGTTTTTTATT
TTAAGTTGGTTATTGTGTGAAGTTGGTTTAGAATTTTTCGGTTTTTGGGAGTTTGTTTTTTCGTTTGTAAAATGGGGTTGTAGGGTCG
TTTACGCGGTTATCGGAAGGATAAGGTGTTTAGGTCGTTAGGTCGTTTTTTGGTAAGCGATTTTTATTCGTAGCGCGGTTTCGATTTTC
GTTTAAGACGCGTTTTTCGCGTTTTTATTTTTTTTAGGTTTTGGTTAGTTTATTTTTCGTTTGGGGCGGTAATTTGTTTTTTTTTGAATT
TTTCGTTTTCGACGGGTTTTTTTTTTTGATTCGCGGTTCGGAGGTTTTTTTTCGTTTTGAAATGTAAATTCGTTTCGGTTGGGGTCGC
GGGCGGTTCGGAGTTATAAAAGGTTTGGGTGGGGCGGGCGCGGCGGTAGGATAGTCGAGTTTAGGTGAGCGGTTGTTCGTCGTCG
GGGCGGTCGGTAGCGGCGGTTTTAGGGTTTAGTATGCGCGGGGGATTTCGCGGT

the primers are :
sense   TTATTTTTTTTAGGTTTTGGTTAGTT
antisense  CCACCCAAACCTTTTATAACTC
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?

[pcrman]

Everything looks fine to me. How many cycles do you run your PCR? You can run 40 cycles, and then take 1-2 ul of the first pcr product as template to run another PCR for 25-30 cycles using the same primers.

[vilperte]

I'm using the PfuTurbo Cx Hotsart DNA Polymerase, it works very well!

You can try to decrease the extension temperature to 65C and increase the time for 2 minutes. It really helped me