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| Epigenetics Forum: DNA Methylation, Histone and Chromatin Study An Epigenetics Forum Dealing with DNA Methylation, the study of histones and chromatin |
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#1
11-06-2008, 05:37 PM
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 bisulfite + taqman- hellpppp... i'm trying to perform bisulfite conversion and real time PCR afterwards in order to find out methylation of BRCA1 gene. im using cell lines of lung and breast cancer and convert only 1 ug of extracted DNA of each cell line, then im doing real time PCR using primers and prob of ALU gene (for start, just to see if i have enough DNA) . im using the taqman for the real time... results of the real time: 2 times i got nothing and 2 other times i got amplification but the CT was too high... can anybody help? pls if you have any experience with this I would be grateful...Im pretty desperate... thanx! (sorry if I have any english mistakes...)  |
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#2
11-14-2008, 09:29 PM
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| Re: bisulfite + taqman- hellpppp... Hi, I did few experiments with bisulfite for methylation studies but only using regular PCR and cloning or pyrosequencing(by a compagny). Did you try regular PCR? 1ug of genomic DNA is a good amount to work with. Are the primers you are using have been used before? |
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#3
11-15-2008, 05:13 PM
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| Re: bisulfite + taqman- hellpppp... yep... the primers and prob are working for sure... I dont know what I am doing wrong. can you give more details about the regular PCR that you are doing? how it helps you for detecting the % methylation? |
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| bisulfite , hellpppp , taqman |
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