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Bisulfite sequencing PCR problem


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#1 utpal

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Posted 23 November 2004 - 05:46 PM

Hi,
I am trying to study the methylation status by bisulfite sequencing in genomic DNA from cell lines. I started with 0.5 ug of genomic DNA and after bisulfite modification followed by purification (QIAGEN PCR purification kit), desulfonation, neutralization (with NH4CH3COOH) and precipitaion I dissolved the precipitate in 10 ul. Out of 10, I use 4 ul for PCR amplification.
I used gradient PCR to anneal at different temp (ranging from 48 to 55 C, Tm of the primers are 60 C). After 35 cycle amplification with ThermalAce DNA pol (Invitrogen), I had no detectable product. The primers were designed by using MethPrimer (www.urogene.org/methprimer/index1.html).

May I request your help.

utpal

#2 pcrman

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Posted 23 November 2004 - 05:59 PM

Hi utpal,

There are some things in your protocol that may be problematic.

1) I am not sure if qiagen PCR purifiation kit can be used for this purpose and have never heard of it. Try some other methods such as silico.

2) You can increase your cycles to 40 or more, alternatively, run two rounds of PCR, first 40 cycle, then reamplify using 25-30 cycles. After the first round, you may not be able to see the product on gel.

3) Hot start your PCR by using some hotstart taq such as JumpStart taq from sigma.

Good luck.

#3 utpal

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Posted 24 November 2004 - 02:12 PM

Hi pcrman,

Thanks a lot for your suggestions.

I tried for reamplification (35 cycles) and still there was no product. However I would go for hot-start Taq pol. Could you please suggest the product name and supplier of DNA purification system of your choice, preferentially spin column based. I would like to try that as well.

utpal

#4 pcrman

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Posted 29 November 2004 - 08:03 PM

I use the Chemicon kit which has the purification reagents included. In my hand, DNA recovery is good and stable.




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