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