Posted 24 October 2011 - 08:37 PM
I am new to PCR and facing a serious problem with my Reverse transcription-Nested PCR reaction.
I am following a Protocol from an authentic published paper, but unable to get desiered PCR product.
The Required Product size for my reaction is 1500bp, but I always get a product of around 250-300bp. Even I have re-ynthesized my Primers and tried different polymerases but no results. Even varying conc. of MgSo4 didn't work.
The thermal profile is touchdown with increasing extension time of 20 sec at each cycle.
I hope I will get the solution from you people .
Posted 25 October 2011 - 06:07 PM
Have you looked at the specificity of your primers? Try primer-blast on the NCBI website, if you haven't.
Small products will amplify more efficiently than bigger ones, if there is a potential smaller product in your mix, it will be amplified in preference over bigger ones.
Which stage of the nested PCR is causing the problem?
Posted 27 October 2011 - 10:15 PM
I'm using the same DNA and exactly the same experiment.
yes the primers are specific n checked in primer blast.
I am using Reverse primer of reaction to synthesise cDNA to avoid non specific bands but still not getting my required product.
the 2nd round of nested pcr is actualy causing problem.
Posted 28 October 2011 - 02:30 PM
Have you optimised the annealing temperatures for your PCR machine - they can vary a bit based on the machine used.
Posted 03 November 2011 - 02:10 AM
I have used that for cDNA synthesis aswel
yes I am trying different annealing temperatures now days but not getting required results still ........
Posted 03 November 2011 - 02:59 PM
Posted 07 November 2011 - 10:05 AM
Edited by merlav, 07 November 2011 - 10:06 AM.
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