PCR with long and complex primers
Posted 25 June 2010 - 06:26 AM
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!!!
Posted 27 June 2010 - 03:44 PM
Is there anything unusual about the template?
Posted 27 June 2010 - 04:17 PM