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Troubhooting PCR

Troubhooting PCR - PCR - Polymerase Chain Reaction Forum

Troubhooting PCR - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 03-07-2013, 04:12 PM
Pipette Filler
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Default Troubhooting PCR



Dear Fellow
Me is working on the project in which amplification of SARM ( CDS portion, 2200bp) is needed. I have made the cDNA and designed the primers with additional restriction sites. But I am unable to amplify.

I am using LA taq

primers
Forward

CAGATCT Atg gtc ctg acg ctg ctt ctc tcc (31 bp)

REverse

GGGTACC Tta ggt tgg acc cat ggg tgc agc (31 bp)

Red colored are the restriction enzyme sites added by me which are not present in my sequence

I have tried below mentioned thermocycling conditions

initial denaturation 95C for 5min
denaturation 95C for 1min
annealing 51 or 53 or 55 or 57 for 1 min
extention 72 for 2 min
and final extenion 72 for 5min
total cylces were 35

Can you please suggest anything ?
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