smears in PCR products (bisfulite treated DNA) - (May/12/2011 )

Hi Everyone,

I've successfully amplified my products with:

Zymo research's "human methylated DNA".

However, when I extract DNA from cell culture, I don't see any products- only smears. I've designed my primers using methylprimer express.

I do 40 cycles.

What does this mean?

Thanks.

You got expected products using control templates, suggesting the primers work. Make sure your DNA quality (purity) and recovery are good. You may also try hot start taq polymerase such as JumpStart redTaq from sigma.

pcrman on Sun May 15 16:56:39 2011 said:

Hi All, I was new to this topic and i was trying to work on nested PCR for a gene which is of interest. As per the literature the bisulfite convert DNA was used as an starting material for the the first step pcr ( methylation-insensitive) and it should amplify between bp 329 and 399 from the transcription site but for me i had got the amplification at bp 70 . I would be grate if any of you can help me to troubleshoot the problem. Thanking in advance

Hi all,

It often helps to keep your forward and reverse primers in seperate stocks.

Also invest a lot in QC. Make sure your cell DNA is very clean and there should not be amplification on plain genomic DNA (yes you test your BS-PCR on gDNA as well: do you have a product, than dont use, as with difficult bs conversions you will most likely get a specific products in your "BS-PCR", which might not be as "BS" as you liked...)

I also had problems with gDNA from fibroblast cultures behaving badly in bisulfite conversions.

It helped to do old fashioned 25:24:1 purifications of the DNA.

If your cell culture DNA bisulfite conversions with subsequent BS-PCRs keep on performing badly switch to a different type of membrane. Somehow it helped to switch to Qiagen's Epitect FFPE protocol

Also, always test your primers thoroughly! And your BS conversions!

best