PCR with degenerate primer - (Dec/27/2005 )

Hi,

I have designed a primer sets which contain some ambiguous nucleotides, such as R, Y, W, K...etc within the primer sequences . I would like to know how should I estimate the annealing temperature for this type of primers?
I have been actually trying to run a few PCRs with different annealing temperature, from 45-55C, but all reactions were failed, and not even a single primer dimer was amplified. Could anybody give me an idea about the how this problem ocurred?
Thank you very much in advance.

Angel

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How long are your primers?

Have you tried using betaine or DMSO?

What is the size and GC content of your amplicon?

-Matt

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Hi, Matt.

My primers are 17bp-long. Do you think I should design a longer primers?

GC content of the primer or amplicon, you meant?

The amplicon size is small, only 200+bp. I see no reason I am not getting it.

Anyway thanks for your suggestion. I will try again later. Please post me any other suggestions if possible, appreciate it a lot.

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Dear all,

I read that the degeneracy of the primers should not be more than 512-fold. Is that because of my primers have a even higher degeneracy, that's why I couldn't get my PCR product?
I am going to try using 500pmol of primer per reaction. Would this amount be too high?
Your suggestion is highly appreciated.
Thanks.

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The Tm of the primers will not change depending on the degeneracy, although, because it is a mixture, some with low GC content will have lower Tm than those with high GC content.

500 pmol sounds like too much primer. Our standard is more like 20 pmol in a 20 ul reaction. Unless you are running very large reaction volumes, 500 pmol is way too much.

I'd repeat an earlier question: what is the length and what are the properties of the template? GC content? Low or high GC areas?

For degenerate primers, it matters most that you have good matches on the 3' end of the primers. Try to locate highly conserved protein regions which use unique codons. Met (ATG) or Trp (TGG) are especially good for this.

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Hi, friends.

After a long holidays, finally I got my amplification products, by using 10pmol of the Forward primers and 50pmol of the Reverse, at a very high concentration of MgCl2 (5mM).

Sigh...

However when I want to use the same degenerate primers for the RT-PCR, it was failed. cDNA could not be formed well by this primers, and PCR products were blank.

Anyone has an idea on how to improve the situation? I could not change the primer since I have to amplify only this particular region of those genes...

Any suggestion would be truely appreciated!!!

Thank you.

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