6 replies to this topic

[de ifz]

Hi,

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 .

Thanks alot

[bob1]

Are you doing exactly the same experiment (same species DNA and everything) as the one in the paper?

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?

[de ifz]

yes bob
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.

[bob1]

Hmm, reverse primer for which PCR step? It could well be that you have some carry-over contamination of something sitting like another primer somewhere in your reactions. This could cause mis-priming or compete with the proper primer.

Have you optimised the annealing temperatures for your PCR machine - they can vary a bit based on the machine used.

[de ifz]

There is only 1 primer for both rounds of Nested PCR reaction.
I have used that for cDNA synthesis aswel

yes I am trying different annealing temperatures now days but not getting required results still ........

[bob1]

Have you tried to make your cDNA by oligo-dT or random priming? It could well be that there is some carry-over of incomplete products between the reactions when you are using the same primer for all.

[merlav]

What are the thermocycle conditions??? could you please write the temp and time? , cycle number and enzyme name

Edited by merlav, 07 November 2011 - 10:06 AM.