Mysterious PCR Contamination - (Sep/02/2009 )

From what I had seen here, perhaps the question arise should be why there are smears in the negative, which is a no DNA template control. How long is your primer anyway?

Try other manufacturer brand's taq. Borrow from somebody to see whether is the Taq's problem, who knows? I experienced failed, unspecific and wrong binding amplification from a locally manufacture Taq but when I switch to a more well establish company's taq, the problem solved.


P/s: forgive me if I ask: why your gel appear to be so "orange" in colour?

Loading buffer is not likely contaminated as the problem shows up only in amplified product. Loading buffer was not used to reconstitute the primer set currently being used, so there is no chance for contaminant in the loading buffer to make it into the amplifications.

The orange color is just the color Ethidium Bromide fluoresces normally.

Swanny, I will try touchdown PCR and see what happens. I have to figure out the highest annealing temp, since my optimized temp is already rather high (58). I probably won't have a chance to run it until next Monday, but I will let you know the result.
I have tried other peoples Taq, both different stocks and different brands.

I am probably going to order fresh primers soon.

Thanks again for everyones input.

One other thing I thought of is running the primers (fwd and rev) without template and the primers asymmetrically. (fwd without template and rev without template).

Next run the PCR with only the fwd or the rev (this time include template). You might also try asymmetrical PCR with template.

Things to try while waiting for new primers.

In my experience, I also ordered new primers with no luck. I still got the smear. The touchdown PCR suggested by Swanny is also a good idea...

Good luck

Start your touchdown 5 degrees above the Tm of your primers. It doesn't really matter if you don't get any binding in the first few cycles. The first primers to bind will be 100% complementary, and after only a few rounds of amplification the correct product will preferentially amplify over all other sequences.