Help needed urgently

[Nita Rajakumar]

Hello,
I am currently working on amplifying a gene by nested PCR. The expected size in the first cycle is 1003bp and in the second cycle it is 483 bp. I was getting good results earlier but now am getting only smears with samples that were positive earlier. I have tried changing the reagents and also doing the pcr with fresh DNA, but no results.

I want to know if a gradient of annealing temperatures would help to optimise it again .I really do not have any more options . There was another doubt in my mind - Will amplification be seen after the first cycle in a nested PCR?

Desperately yours,
Nita

[danfive]

Will amplification be seen after the first cycle in a nested PCR?

Can be either Yes or No--depends on the amount of target DNA in the sample, some don't become visible amounts until amplification by nested PCR.

I would repeat with the freshest DNA you have, make sure the thermocycle programs are all entered correctly, and primers are also fresh working solutions, might want to add a tiny extra amount of Taq as well.

[danfive]

Just noticed you mentioned smears--that can be RNA (smears are in the lane not necessarily the area of PCR product) rRNA is the primary culprit in smears, RNAse step or dilution helps) or DNA degradation in sample.

[Nita Rajakumar]

Dear Dan,
Thanks for the suggestions. Will repeat with fresh samples.

Nita

[Nita Rajakumar]

Thanks Danfive. I have checked for the quality of genomic DNA which was seen as a bright band of high molecular weight with no smears in the lane below.Is there a chance that RNA contamination can happen with the reagents tho I have tried changing all the reagents. I will try a dilutuon step too to reduce the smears.

Nita