Help! No bands after BSP! - (Aug/08/2012 )

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?

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.

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

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 ??????