1 reply to this topic

[nyt1983]

Hi everybody,

I'm new in the field of epigenetics and would be very grateful if somebody of this forum could answer my questions

1) After bisulfite treatment, the forward and reverse strand are no longer complementary. Therefore I shoul measure DNA concentration via Nanodrop for ssDNA, right?

2) After BC-PCR I should have dsDNA again, i.e. I can check for a BC-DNA band (or smear via gel electrophoresis using a standard DNA dye containing glycerol, right? Just to verify whether conversion was succesful.

3) I read in some threads that amplicons up to 1kb were amplified! I extract DNA from frozen mouse brain tissue, i.e. brains were taken, frozen and sliced in 200µl slices via a cryocut within 1 week the latest. Specific brain regions were punched using a 1mm diameter needle or whole brain slices were scratched of glass slides. Right after punching or sratching, DNA will be extracted and diluted to 10 ng/µl if possible. I use the Epitect bislufite kit to convert 1µg of DNA into BC-DNA. I try to amplifiy fragments ranging from 150 to 500bp using BC-PCR. Nevertheless almost none of the primers work. I think it is not a problem of primer design. I used BiSearch and spent really a lot of time in designing these primers. Recently I checked my BC-DNA and was quit shocked to see no bands or smear on an agarose gel. I talked to some other PhD students and they gave me the hint to aliquot and store my BC-DNA at -80°C and thaw it just once since the DNA somehow dissapears after freezing and thawing again. Has anybody of you made a similar experience?

4) Right now, I'm going to try my BC-PCR with BC-DNA stored at-80°C using 10 and 20ng, respectively. How much BC-DNA do you use during your PCR?

5) Can you please explain the principle of nested PCR to me?

Thank you very much.

Kind regards.

[RKA]

nyt1983, on Oct 13 2009, 10:29 AM, said:

Hi everybody,

I'm new in the field of epigenetics and would be very grateful if somebody of this forum could answer my questions

1) After bisulfite treatment, the forward and reverse strand are no longer complementary. Therefore I shoul measure DNA concentration via Nanodrop for ssDNA, right?

2) After BC-PCR I should have dsDNA again, i.e. I can check for a BC-DNA band (or smear via gel electrophoresis using a standard DNA dye containing glycerol, right? Just to verify whether conversion was succesful.

3) I read in some threads that amplicons up to 1kb were amplified! I extract DNA from frozen mouse brain tissue, i.e. brains were taken, frozen and sliced in 200µl slices via a cryocut within 1 week the latest. Specific brain regions were punched using a 1mm diameter needle or whole brain slices were scratched of glass slides. Right after punching or sratching, DNA will be extracted and diluted to 10 ng/µl if possible. I use the Epitect bislufite kit to convert 1µg of DNA into BC-DNA. I try to amplifiy fragments ranging from 150 to 500bp using BC-PCR. Nevertheless almost none of the primers work. I think it is not a problem of primer design. I used BiSearch and spent really a lot of time in designing these primers. Recently I checked my BC-DNA and was quit shocked to see no bands or smear on an agarose gel. I talked to some other PhD students and they gave me the hint to aliquot and store my BC-DNA at -80°C and thaw it just once since the DNA somehow dissapears after freezing and thawing again. Has anybody of you made a similar experience?

4) Right now, I'm going to try my BC-PCR with BC-DNA stored at-80°C using 10 and 20ng, respectively. How much BC-DNA do you use during your PCR?

5) Can you please explain the principle of nested PCR to me?

Thank you very much.

Kind regards.

yes it is true that you have to measure the dna con as ssDNA after bs conversion. yes indeed the converted dna is very sensitive and u need to use as aliquots. I have used EZ kit before, worked with FFPE. I have used 800 ng DNA for convertion and appx 20 to 40 ng DNA was used for subsequent PCR. You cal also check the bs conversion with universal primers