Very dense primer-dimer band but no product - (Aug/29/2005 )

The PCR products which I am trying to obtain are ~100-150bp long.

After running it after agarose, there is no band (only very very dense and blurry primer-dimers bands at the bottom)

I have tried many things:
Annealing Temperature: from 45-60C
Magnesium Concentration 1-5mM
Different samples
Different cycles
Different set of primers

There is nothing wrong with my reagents and there is no contamination, because during the experiment - I have to quantify more than 15 genes, approximately seven of them are consistently there, however the other 8 which is well known to be expressed in the tissues are not there!! (All these experiments are done simultaneously)

The products which i can probe don't have these dense primer dimer bands.

But i have check the sequences of my primers and they are not very complementary at all.

What could be wrong??

Help!

Eva

You could have "poisoned" your PCR (ie contaminated your work area, pippetors or solutions with primer dimers). If this is the case the best option is to set up in a different lab using different pippetors with all fresh reagents.

Another thing you may want to try is hot starting all your reaction- this can really reduce primer dimer problems.

Daniel

Molecular biology troubleshooting

try increasing the amount of template!

How much primer are you adding to your reaction? The blurry band could just be excess primer, not necessarily dimers.

I would try to do a temperature gradient PCR to find the optimal annealing temperature (which might be higher than 60 degrees depending on the melting temp of your primers). Generally a range between 55 and 70 degrees is a good idea.



Also which primer design program are you using to determine the complimentary of the primers? Does it take into account self-complementary primers or secondary structures? Different programs also give different results for this prediction.

You are saying the sequences aren't very complementary... so they are to some degree. Based on your results it is my opinion that the problem is based around your primer design.

You could try adding up to 10% DMSO in your PCR to relax potential secondary stuctures in your primers, but really I would check the primers with a good design program first. primer3 is available online or you could use Sigma-Genosys' online calculator. You'll have to sign up for that one but it's free. Just follow the procedure to order primers, but don't click on "Buy". You can find both links through google.