cDNA amplification problem - (Feb/21/2013 )
Long story short... I'm trying to amplify a full length cDNA from mouse forebrain for cloning. I used trizol to extract the mRNA and InVitrogen's Superscript III first strand cDNA synthesis kit to make the cDNA. I used 10% (2 uL of a 20 uL rxn) for the template and I did a temperature gradient for annealing (55, 60 and 65, with the Tm being around 68) using high fidelity polymerase (pfu ultra hotstart). In addition, as controls, I used GoTaq to amplify GAPDH and my full length cDNA at 60C. Once the results came back, none of my high fidelity reactions worked, but I got my full length band and the GAPDH band with GoTaq, suggesting that the cDNA was there. Fearing that this result was due to the polymerase, I then tried it with another high fidelity polymerase (Clontech's Advantage HD), but same result.
I'm baffled on how to continue next. I'm guessing there may be some crap in the cDNA mix that is interfering with the reaction. I was thinking about doing an ethanol precipitation and purifying the cDNA and then trying again. Any of you have any experience with this?
Is it necessary to use high fidelity polymerase? Maybe you can just use Taq and then sequence your cDNA. Taq isn't that bad...
I have experienced the same problem, where I used to get the amplification with the taq polymerase, but with pfu polymerase the gel used to be blank. Try using Advantage DNA polymerase mix from clontech. I found it really useful giving the opposite results. It gave the amplification with the primers which did not work with the normal taq pol.
If all you are doing with the DNA is sequencing it, then Taq is a good choice for a polymerase. Errors it makes will almost never be seen in a sequencing reaction, since the sequencing reaction looks at the consensus base at each position. If you are cloning, then it makes a big difference, since you are selecting a single molecule, which will ofen contain errors.
I've tried the clontech mix already. It didn't work.,,, but an undergrad loaded my sample... so I'll try that again today along with the GAPDH control....
any other suggestions??