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Can anyone help with my Bisulfite PCR?


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#1 newepi

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Posted 23 July 2012 - 11:20 AM

Hi folks,

I am new in the bench work field. I just started working on my BS-PCR, but all the runs were failed.
There can be different reasons which made the BS-PCR not work, for now, what I can think of include:
1) The annealing temperature setting in the thermocycler: the Tm of my Forward primer is 48C and the Tm of my Reverse primer is 66C, the difference in the temperature between the pair of primers is sort of large (18C), is that gonna be a problem? if not, then what temperature would you guys suggest I use? I used 48C for my previous run.
2) There could be some issues with the bisulfite conversion step: I have not done any thing to check if the conversion was complete. And one of my lab mates also told me that if the bisulfite treat was too much, then it could do damage to the DNA, and I did not know anyway to check that either.
3) I used MethylPrimer express to design my BS-PCR primers: It is the first time I design primers in my life~~ (again, I had no wet lab experience before). I did not know if there is anything I need to pay attention to when designing primers, especially for BS-PCR primers.

I appreciate any of your insights here.

Thank you very much,

newepi

#2 pcrman

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Posted 23 July 2012 - 01:00 PM

Hi newepi,
Bisulfite PCR is very different from regular PCR in that the DNA has been degraded during BS modification to a great extent that amplifying it becomes very difficult. Another issue is that primer design on BS sequences is also very challenging.

1. Big differences in Tm of the two primers in the pair are common. you can choose a happy medium for example 55C
2. Partial conversion is an issue sometimes, regardless, you can still amplify those already converted.
3. Two rounds of pcr are usually required to see a band. You can reamplify using the same pair or use nested primers.
4. Hotstart taq polymerase e.g. JumpStart Red Taq from sigma helps your PCR.

#3 newepi

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Posted 25 July 2012 - 10:21 AM

Thank you very much, pcrman!

Hi newepi,
Bisulfite PCR is very different from regular PCR in that the DNA has been degraded during BS modification to a great extent that amplifying it becomes very difficult. Another issue is that primer design on BS sequences is also very challenging.

1. Big differences in Tm of the two primers in the pair are common. you can choose a happy medium for example 55C
2. Partial conversion is an issue sometimes, regardless, you can still amplify those already converted.
3. Two rounds of pcr are usually required to see a band. You can reamplify using the same pair or use nested primers.
4. Hotstart taq polymerase e.g. JumpStart Red Taq from sigma helps your PCR.






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