Problems cloning cDNA using Pfx - Problem (May/24/2005 )

Hello,

If I use Taq polymerase I am able to amplify various cDNA sequences that I have been attempting to get from cDNA synthesized from myeloma cell lines. But everytime I use the same cDNA samples and same primers I am unable to obtain a product using the proofreading Pfx polymerase (Invitrogen). The only success I have had in the past with Pfx is when I have been using a maxiprep of a vector containing the cDNA.

I need to amplify a 2.5kb fragment. I have confirmed cDNA is good by using Taq and also our microarray data shows this cell line has high expression of our gene of interest. All my attempts to date have failed with Pfx. Does the concentration of any of the reagents differ for Pfx from what would be used for a Taq PCR reaction?

Does anyone have any ideas of what may be the problem.

Cheers,

Pete

I posted this the last time. Here it is just in case you missed it.


Here is a journal about the efficiency of different polymerases. Looks like you fragment is too large for pfx.

www.gene-quantification.de/arezi-2003.pdf

This is an expensive fix but this is what worked for me. Double the amount of dntp's, MgSO4, and primers called for in the product protocol. Use the buffer at 2.5X final concentration and 1.0 ul of enzyme. Make sure to only go 30 sec/kb on your extension time. I have generated a 3 KB product with pfx this way.
Goooood luck .