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PCR of bisulfite converted DNA is now producing a smear? Previously produced a s

Bisulfite Conversion PCR Smearing Bisulfite Sequencing

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#1 Epigeneticist

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Posted 24 May 2014 - 02:02 PM

I recently tried to PCR amplify some sodium bisulfite converted DNA samples for bisulfite pyrosequencing. I am using two sets of published primers and they produced a single band for me in the past (one is 143bp and other is 341bp). Not sure if the primers are getting old or if the DNA is not getting fully converted. The primers are about a year old and stocks have been stored at -20C. I was able to get the one set of primers to work by lowering the annealing temperature from 55 to 51C. However,  the other set of primers will not produce a single band. They worked at 59C but now only produce a smear with annealing temperatures from 62-49. 

 

What is the problem?

 

Degraded primers?

 

Incomplete conversion?

 

Contamination?

 

Thanks!

 

 



#2 phage434

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Posted 25 May 2014 - 05:50 AM

Are you working with newly converted DNA templates? Converted DNA is largely single stranded, and quite sensitive to degradation. Other possibilities include PCR inhibitors in your converted DNA. Try reducing the amount of template you add. If you are using a reliable kit (Zymo e.g.) then I doubt it is incomplete conversion. Your primers should be stable, especially if you store them in TE rather than water.



#3 Epigeneticist

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Posted 25 May 2014 - 12:56 PM

Are you working with newly converted DNA templates? Converted DNA is largely single stranded, and quite sensitive to degradation. Other possibilities include PCR inhibitors in your converted DNA. Try reducing the amount of template you add. If you are using a reliable kit (Zymo e.g.) then I doubt it is incomplete conversion. Your primers should be stable, especially if you store them in TE rather than water.

 

Conversion - I always use a zymo kit. All reagents were fresh and incubation was 16hrs. The DNA was eluted from the column and used immediately for PCR. 

 

Primers - stored in water at -20C (in a non-frost free freezer). I actually store all my stock primers in water. Maybe I will store my stock primers in TE. 

 

Primer degradation seems more likely. 

 

Thanks!







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