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DNA bisulphited Amplification


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#1 flowerz

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Posted 08 July 2010 - 02:04 PM

To analyze if the promoters of a gene (in which we are interested) are methylated the genomic DNA was digested.Then, the DNA (2ug) was bisulphited following a standard protocol (16 hours at 55 C). The bisulphited DNA was cleaned, precipitated, purified and re-dissolved in 20 ul on Buffer TE. A PCR was performed with primers that had been designed with the program "Meth primers". But 0 product!!.
Then, we tried to digest or not the DNA, used a different Sodium Bisulphite, changed the PCR's conditions, but nothing happened!. Moreover, the DNA and Magnesium Chloride concentration were modified, and the primers and enzymes were changed. Finally, a nested PCR (using different cycling programs) was tried. But there is no band!
Any help is welcome,
Thanks!!!

#2 pcrman

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Posted 08 July 2010 - 03:29 PM

Please give your PCR conditions and tell us how you digested your DNA.

#3 methylnick

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Posted 08 July 2010 - 04:42 PM

Further to PCRman's comments, are you using primers that you know work in the past? or are they newly designed?

post the sequences too

#4 flowerz

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Posted 09 July 2010 - 05:26 AM

Thank you for the answers!
Yes, Initially the primers were working! But now they began not to walk. We checked the sequences a lot of times!
With the traditional PCR (three steps: denaturalization, annealing and extension) not band was detected, therefore we designed one of only two steps: denaturalization to 80C 15 seconds and annealing + extension to 60 C 60 seconds. Every was very well with this cycle, but only hard a time ...! We know that the DNA is unstable, so inclusive we have proved it with DNA newly bisulphited.
With regard to the cut, we follow the instructions of the specification sheet of the enzima, know that the domestic purification might be a point in against.

Forgive my Englishman's mistakes! And Thanks again!!




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