Large smear in PCR - (Jul/17/2008 )
I am using iProof high fidelity DNA polymerase(BioRad) to amplify 1.9kb of MYO7A (mouse).
I set up 20ul reaction tube and everything was working well, I got the band of interest , optimized the PCR condition and ordered new polymerase and dNTP to go for a large scale amplification(x100ul tube). After receiving the order and doing the PCR reaction I get a large smear after the band of interest (smaller 1.9 kb). I repeat the reaction using different reagent (new stock v.s old stock) and change the water , tube and everything that I thought could be source of contamination, I also made new stock of primer; I am getting the same smear. I also try changing the cycle number from 30 to 25 and 40, same problem plus I dont get the any amplification of band of interest.
What surprises me is, it was working perfectly (first 4 trial) and then from a point I cant get the same result. I have negative control in all trial and I get the same band with the same trend in each and every PCR. The intensity of the band is also less than the first time I tried that.
I can do the gel purification but still I am getting less yield and I know that it can be fixed, but how???
I appreciate any advise,
My guess: the smear is likely primer dimers or some other extraneous DNA that came with your DNA polymerase. You could use a very clean DNA polymerase (if you can find one), or just use a Hot Start style DNA polymerase from your vendor of preference. It doesn't appear that iProof has a hot start formula.
Could you post a photo of the gel, reagents concentrations and PCR conditions so it will be easier to find the problem.