PCR with long and complex primers - (Jun/25/2010 )

Hi to everybody!

I have tried to do a PCR for more than one month and now, after so many hopeless attempts, I have decided to ask your opinion… because maybe I am doing something wrong.
It is not a standard PCR because I am using quite long primers (70 bp) which should anneal to a plasmid template with their last (3 prime) 20 bases. The primers contain secondary structures and are prone to produce dimers… however I cannot design them in a different way. In addition, as you can imagine, due to their length they have very high Tm (>80°C). The product I am expecting is around 1,5 kbp.

Here the reaction set up:
- Around 50 ng plasmid
- 2 uM of each primer

After an initial denaturation at 94° for 3 minutes the following cycle starts:
- 94°C for 30 seconds
- 52°C for 30 seconds
- 72°C for 2 minutes ) x 30 times

My problem is that I do not get absolutely any PCR product (as analyzed by gel electrophoresis). From time to time it happened that I was obtaining some very low intensity bands at the expected size… but they were really weak.

Is there anything I should do or try to increase the chance to obtain the right PCR product? Just as a general information, I am doing other PCR in parallel and they all work fine… so I think the problem is linked to this specific PCR.

Let me thank you in advance for your help!!!

Have you tried additives like DMSO or betaine? They will disrupt secondary structures, in both the template and the primers.

Is there anything unusual about the template?

For this PCR the Tm of the primers is calculated using the 20 bp that are specific for binding to the template, ignore the rest for the time being. I also second adding a 2ndary structure inhibitor such as DMSO or betaine.