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Bisulfite Sequencing and PCR Troubleshooting

Bisulfite Sequencing PCR Primer Design DNA Methylation Epigenetics

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#1 5280



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Posted 18 November 2013 - 07:47 PM

I designed some primers on MethPrimer to target my gene of interest. I have previously optimized primers for bisulfite converted DNA so this is the first time designing and optimizing them. Above 59C no product, 59-57 very faint band of correct size, and below 57 has very strong primer dimer bands. I have repeated the gradient with hot start taq polymerase and it doesn't help. Tried 40 and 45 cycles of PCR, and varying input amount of bisulfite converted DNA. The band is so weak that I am not able to excise for gel purification. I have a considerable amount of experience optimizing PCR conditions just do not know if the primer design is bad. 


Any recommendations for enhancing product or trying to get rid of primer dimers? I have fought with primer dimers in the past but my band of interest was strong enough I could gel purify. 


Primer redesign?


Any general recommendations for primer design?


Amplicon size? Mine is 700bp


Tm? These are 59 and 60C. 





#2 phage434



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Posted 18 November 2013 - 08:52 PM

Since you seem to have some product, I'd recommend designing a nested primer and use it to amplify your low-abundance product. I'd probably design two, one at each end, and try them separately (with one of your existing primers) and together.


You could also try lowering your extension (not annealing) temperature. The very high AT content of converted DNA makes a lower extension temperture useful. Try 63-65 degrees, with a bit longer extension time.

#3 pcrman



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Posted 19 November 2013 - 06:17 PM

Amplicon size? Mine is 700bp


You really need to reduce the size of your product to something < 400 bp, reason being DNA is greatly degraded during bisulfite treatment. 


Other tips: Try different primer sets, try nested PCR (first round 40 times, 2nd 30 times). Hotstart is necessary. 

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