molecnewb

Hi,

I've been using this forum quite extensively in the past few months to try to figure out BSP but now I have hit a roadblock...I have been trying to amplify part of a CpG island in a gene and it had worked initially, albeit not too well, but I was still able to get some sort of band. I used MethylNick's protocol and was able to get a band but when we Sanger sequenced it, there were a lot of erroneous reads and many G's that did not match at all to the template sequence. We tried to use a regular PCR protocol (standard, 30s denat, 30s, anneal and 1 min elongate) and was able to get a good band for a while. Much stronger and very straight forward. However, lately, in the past few weeks, I am unable to get this band again. Instead, I have some laddering that could be primer dimerization (?) amplifying on itself and it does not correspond with the product size at all. I have tried to make a new working solution of primers, new bisulfite DNA, Taq has recently been bought but nothing worked. I'm still getting this laddering effect for all of my samples. I am using 5% DMSO and Qiagen Hotstar Taq as well after bisulfite conversion with Epitect.

Original sequence:
GGCGGAGGCGCAGGCGGTGGCGAGTGGGTGAGTGAGGAGGCGGCATCCTGGCGGGTGGCTGTTTGGGGTTCGGCTGCCGGGAAGAGGCGCGGGTAGAAGCGGGGGCTCTC
CTCAGAGCTCGACGCATTTTTACTTTCCCTCTCATTTCTCTGACCGAAGCTGGGTGTCGGGCTTTCGCCTCTAGCGACTGGTGGAATTGCCTGCATCCGGGCCCCGGGCTTCCCG
GCGGCGGCGGCGGCGGCGGCGGCGCAGGGACAAGGGATGGGGATCTGGCCTCTTCCTTGCTTTCCCGCCCTCAGTACCCGAGCTGTCTCCTTCCCGGGGACCCGCTGGGAGC
GCTGCCGCTGCGGGCTCGAGAAAAGGGAGCCTCGGGTACTGAGAGGCCT CGCCTGGGGGAAGGCCGGAGG

Bisulfite treated sequence:
GGTGGAGGTGTAGGTGGTGGTGAGTGGGTGAGTGAGGAGGTGGTATTTTGGTGGGTGGTTGTTTGGGGTTTGGTTGTTGGGAAGAGGTGTGGGTAGAAGTGGGGGTTTTTT
TTAGAGTTTGATGTATTTTTATTTTTTTTTTTATTTTTTTGATTGAAGTTGGGTGTTGGGTTTTTGTTTTTAGTGATTGGTGGAATTGTTTGTATTTGGGTTTTGGGTTTTTTG
GTGGTGGTGGTGGTGGTGGTGGTGTAGGGATAAGGGATGGGGATTTGGTTTTTTTTTTGTTTTTTTGTTTTTAGTATTTGAGTTGTTTTTTTTTTGGGGATTTGTTGGGAGT
GTTGTTGTTGTGGGTTTGAGAAAAGGGAGTTTTGGGTATTGAGAGGTTT TGTTTGGGGGAAGGTTGGAGGGTGGGTGGTGT

Forward primer:
GTGAGTGAGGAGGTGGTATTTT

Reverse primer:
AAACCTCTCAATACCCAAAACT

Any help would be greatly appreciated! Thank you so much!!

-molecnewb