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PCR cloning, faint band - (Jan/30/2007 )

Hi,
I am doing a PCR amplification of the gene of interest which is 990bp long. The limitation is the cDNA which I have borrowed and the cells/RNA/cDNA is difficult to avail. Over that the concern is I get the expected band but it is too faint. I did a 50ul reaction, using Finnzymes' Dynazyme PCR master mix( 0.04 U/µl DNA Polymerase, 2x optimized Buffer and 400 µM of each dNTP. The master mix supplies 1.5 mM MgCl2 in final reaction concentration. ).PCR for 40cycles with 10' extension step. I ran the whole sample on gel still band is invisible directly on a transilluminator and can be seen only on gel doc system. I tried using this product as template for another set of PCR which did not give any amplification! Is there any way I can get an intense band with whatever cDNA I have.
Thank you

-Calvin*-

Calvin, can you decrease the annealing temperature? what are the Tm of you primers? also try to increase the amount of the template. Good luck!

-Kathy-

I am also facing similar problem with my PCR with Finnzyme Phusion DNA polymerase, though I got my expected PCR product. It was still very faint after I loaded in everything in the well for electrophoresis (the primer dimers sometimes even look brighter than the PCR product).
I have done gradient PCR and extended the extension time to 1 minute instead of 30 sec.
One of the problems I am facing at the moment is I have very limited cDNA, which was given by another lab, and the cDNA concentration is unknown.
I am thinking of gel purified the PCR product and perform another round of PCR from the gel purified product.
Please give me some suggestions for this problem. Many thanks. smile.gif

-virus_fan-

In both cases, a nested PCR reaction would be ideal if you have information about the internal sequence. Reamplfying with the same primers rarely works. Even a few bases different at the 3' end can make a second round nexted PCR or semi-nested PCR reaction work well.

-phage434-

QUOTE (phage434 @ Feb 3 2007, 08:26 AM)
In both cases, a nested PCR reaction would be ideal if you have information about the internal sequence. Reamplfying with the same primers rarely works. Even a few bases different at the 3' end can make a second round nexted PCR or semi-nested PCR reaction work well.


Samples likely have residual dUTP which is inhibiting your proof-reading polymerases. Taq does not stall from dUTP.

Purify further, increase the concentration of your template, and use a taq/vent mixture (about 2 U taq/0.02 U vent) with glycerol/DMSO.

-Matt

-MisticMatt-