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PCR with long and complex primers


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#1 philip83

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Posted 25 June 2010 - 06:26 AM

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!!!

#2 swanny

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Posted 27 June 2010 - 03:44 PM

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?
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#3 bob1

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Posted 27 June 2010 - 04:17 PM

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.




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