Pfx PCR problems - (May/19/2005 )
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 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 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 anyone have any ideas of what may be the problem.
Cheers,
Pete
-myeloma_pete-
QUOTE (myeloma_pete @ May 19 2005, 05:09 PM)
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 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 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 anyone have any ideas of what may be the problem.
Cheers,
Pete
If I use Taq polymerase I am able to amplify various cDNA sequences that I have been attempting to get from cDNA synthesized from 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 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 anyone have any ideas of what may be the problem.
Cheers,
Pete
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
-dobbiewalton-