PCR problem..... - (Oct/03/2007 )

Hi to all
I am trying to clone certain genes out of cDNA.
I have designed primers prior to start and stop codon with high GC rich content. My expected band size is 4 kb
After running PCR reactions with this genespecific primer pairs on my cDNA I get 3 bands with low size ( 1kb,0.8 kb, 0.5 kb),
I have also tried Rt-PCR with Primer STAR DNA POL....but here also I couldnt get specific band...
Please let me know about your experiences, tricks and techniques.

Thanks In advance.......
All d best

Santosh

Hi! I have not any experiecce of my own, but another in the lab had that problem. She got a product that was missing a large part in a GC rich area. She used Advantage GC cDNA PCR kit from Clontech and had the GC melt both in the cDNA synthesis and in PCR (where polymerase and the GC buffer in the kit was used). and this solved her problem.

I have also read on internet that you can create this GC melt your self in a cheeper way but I do not remember how...

I have found that adding betaine to my PCR helped a lot with amplifying amplicons with high GC content and secondary structure. You will find that a lot of kits for GC templates will contain betaine but they wont tell you that...!

You can also try another primer pair. I had a similar problem with a 3kb fragment and the first primers I had designed just didn't produce the right band. I designed another pair and it worked just fine.

How long are the primers, and what is the Tm? Try to make it ~65C, even if it means your primer is only a 15mer.

Betaine is a really good suggestion.