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DNA methylated PCR


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#1 milla

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Posted 28 September 2009 - 08:49 AM

Hi everybody,

I need some help! I am trying to do a methylation analysis of my promoter.

First I did the modification of the DNA by using the DNA modification kit (S7820) from Chemicon, after that I am trying to PCR my fragment in order to sequencing it.

Unfortunately my PCR is giving any results, even after many different attemps (the fragment is less then 500 bp and i designed my primers by using MSPPrimer).

I know that the problem could be in the primers, but there is any way to see if the methylation works fine?

Any suggestions?

Thank you very much!

#2 rafel61

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Posted 07 October 2009 - 08:03 AM

Hi... I have the same problem. I donīt believe that the primers are wrong. In my case, I figure out that the problem could be the bisulfite modification kit. A person in my lab used the Chemicon Kit and the problem was that the DNA modification was incomplete, or the DNA was looseed in the bisulfite reaction. I recomed you to do the modification procedure without the kit. You could try with the Imprint DNA kit from Sigma, but Iīm not sure that Sigma Kit works pretty well..

Hi everybody,

I need some help! I am trying to do a methylation analysis of my promoter.

First I did the modification of the DNA by using the DNA modification kit (S7820) from Chemicon, after that I am trying to PCR my fragment in order to sequencing it.

Unfortunately my PCR is giving any results, even after many different attemps (the fragment is less then 500 bp and i designed my primers by using MSPPrimer).

I know that the problem could be in the primers, but there is any way to see if the methylation works fine?

Any suggestions?

Thank you very much!



#3 gangut

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Posted 10 October 2009 - 01:04 PM

Hi,

it actually happens quite often that pcr gives no results.. converted DNA is really a difficult template. I guess you tried gradient PCR but did you also try lowering elongation temperature? 68 deg sometimes works better. Did you use taq polymerase?

to check how your DNA is doing after convertion you can try to measure it using spectrofotometer, for ex NanoDrop. Parametres of such dna are generally bad but at least you'll see that it is still there, not completely sheared

One thing you could do to check how successful the conversion itself was, is to run pcr using primers you use for standard pcr of any gene - hostgene for example. Choose primers that have Cs on 3' end that is crucial for annealing. If conversion was successful, primers shouldn't anneal (as Cs in DNA are Ts now), or at least anneal to only a few DNA strands that were not succesfully converted and so there should be no product. If you run normal PCR, run not more than 35 cycles to avoid artifacts. This can give you an answer .. sometimes... as it happens of course that primers work even when they theoretically shouldn't, moreover, T easily anneals to anything, etc, etc :huh: it's a hard life

I use sigma imprint mentioned by Rafel61, it's ok but it's the only one i used so i can't compare it to anything else




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