cDNA amplification problem
Posted 21 February 2013 - 10:29 PM
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?
Posted 22 February 2013 - 02:17 AM
Posted 22 February 2013 - 03:14 AM
Edited by rmbio, 22 February 2013 - 03:16 AM.
Posted 22 February 2013 - 06:18 AM
Posted 22 February 2013 - 07:06 AM
any other suggestions??