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repeat PCR amplification problem - (Dec/06/2005 )

Hi everyone,

I had to amplify a product of 2kb from genomic dna. genomic dna was column purified and on PCR got a weak amplification. I diluted the rest of 10 microlitre PCR product in 500 microlitre of nuclease free water.

because the ampicon is weak, I tried to enrich it by doing repeat PCR using 1 or 2 microlitre of diluted product. I could not see any band, but I see very good amplification with the same forward primer and an internal primer or with the same reverse primer with an internal forward primer. The sizes are of expected size. Amplification using internal primers show that the initial weak band is a specific amplification.

I am unable to understand why I am not able to amplify the entire fragment when the same fwd and rev primers are working with another set of internal primers.

I use Accu Taa LA polymerase from Sigma with its buffer (2.5mM Mgcl2, 2% DMSO).

Thanks in advance for any suggestions.

-rpsrk-

I guess you have used enough extension time for the 2kb PCR.

This is kind of strange. A coworker of mine is currently having the same problem. He is trying to amplify 1.6kb cDNA without success but he can amplify shorter regions with internal primers.

You can try two step PCR: 94C for 30"-1', then 68-72C for 2-3'.

-pcrman-

QUOTE (pcrman @ Dec 6 2005, 05:58 PM)
I guess you have used enough extension time for the 2kb PCR.

This is kind of strange. A coworker of mine is currently having the same problem. He is trying to amplify 1.6kb cDNA without success but he can amplify shorter regions with internal primers.

You can try two step PCR: 94C for 30"-1', then 68-72C for 2-3'.



Hi pcrman,

Thanks for ur reply.

Infact I did a touchdown to get the initial amplification.
The repeat PCR was done with this weak amplification from touchdown. Once I tried to do with 55┬░ annealing and also also repeated touchdown with diluted amplicon as a template (from 68 to 55 with 2┬░decrement), but no result.

The extension time i used was 5 min.... I am unable to understand how and why the same primers work with another set of internal primers and not within themselves.

Any suggestions are welcome

-rpsrk-