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PCR primer usage (Clonning & cDNA)


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#1 Wilson0815

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Posted 12 March 2013 - 09:12 PM

Dear all,

I am trying to optimize PCR. The problem I'm facing now is that while trying to optimize one of the primers, no PCR product is seen.
As I am using PCR primers from previous researcher and he used it for clonning purposes and the primer set actually has an additional fragment(6 bp) at both (forward & reverse) 5' ends.
My work now aims to amplify from the cDNA that I obtained from RT.

The suggested temperature done by previous researcher is 56 degree celcius but I've tried gradient PCR ranging from 54 to 60 degree celcius but still no product is seen.
* At the same time, I also did gradient PCR for other primers and they worked well. I also used master mix so I think it has nothing to do with the reagents am I right?

I'm wondering if the additional fragment kind of interferes with the PCR so the primers cannot anneal in the first place. Any ideas?

Thank you everyone in advance!

#2 bob1

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Posted 13 March 2013 - 12:24 AM

Tailed PCR is common, 6 bp shouldn't interfere too much with it, unless it creates a hairpin in the primer sequence.

If you are using the former researcher's primers, re-order the stocks ( modify if you want), it could well be that they are degraded.

#3 DrRupesh Kumar Rajput

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Posted 13 March 2013 - 01:45 AM

You can use Tm 51 degree celcius.
And an initial set 42 degree for 5 cycles and then 51 degree for 35 cycles.
In initial 5 cycles, extension time will be less, better to keep extension for 20-30 seconds only.
You go with this, i am sure you will get the amplification.
Good luck


Dr. Rupesh Kumar Singh
PhD (Plant Biotechnology)
Department of Biotechnology
Kumaun University, Nainital,
Uttarakhand, India-263139
Mob- +91-9911896375




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