Help! Does anybody use Imprint Modyfication Kit from Sigma?? - (Sep/07/2009 )

Hello everybody

I'm fairly new to DNA methylation analisys and I have a big problem. I use Imprint Modyfication Kit from Sigma.
The question: is there anybody use or used this kit?? What kind of (from where) polimerase, buffer, dntps and concentration do you use for PCR reaction?
I want to make direct sequencing of PCR- product. Is it possible to make sequencing without cloning after I use this Imprint Kit?
Please help me - because I have no chance to change this kit to another

Hi, I use Sigma Imprint, can't tell if it's perfect but it works

I don't think source of polymerase etc matters, it's all about PCR conditions. try to use normal taq however as converted dna is a difficult template and the proofreading activity of pfu doesn't help to handle it. I heard the ABi AmpliTaq works well but never used it

as for direct sequencing, it depends on the number of your potentially methylated Cs and amount of primer dimers. In my case it worked a few times but in general there was too much noise for the sequence to be readable. Anyway, it depends more on the PCR than conversion itself

RedRosa on Sep 7 2009, 03:25 AM said:

Hello everybody

I'm fairly new to DNA methylation analisys and I have a big problem. I use Imprint Modyfication Kit from Sigma.
The question: is there anybody use or used this kit?? What kind of (from where) polimerase, buffer, dntps and concentration do you use for PCR reaction?
I want to make direct sequencing of PCR- product. Is it possible to make sequencing without cloning after I use this Imprint Kit?
Please help me - because I have no chance to change this kit to another

Thank you Gangut

I think it is not a hopeless case, but right now I don't know what is wrong?