PCR Contamination issues - PCR false positives (Oct/01/2004 )
ghezal
Is it possible that you have accidentally introduced reverse transcriptase into some of your stock reagents? I have had similar problems myself recently which are yet to be resolved !!! (after months of the RT-PCR working really nicely), and then somebody in the lab had this problem and now 3 of us have it! I know that these bands cannot be genomic as they are the same size. Also, like you I think, my negative control for the PCR reaction is always negative, so presumably, if cDNA is not the problem (I use the same pipettes with filter tips for the RT reaction and adding the cDNA to the tubes, but diff pipettes for making master reaction mixes), then somehow RT is the problem???
Hi there,
I have a problem with my PCR.I am trying to clone a 3 kb fragment from chicken brain cDNA.I have 3 set of primers and lots of combinations.I tried 2 of them.When I use the combination (F1-R3) that covers all the desired fragment, I saw a smear both with my template and negative control but when I use another internal primer pair (F1-R2)I could not see anything both with my template and my negative control.All PCR reagents that I use for these 2 PCR are same except primers.So I think may be there is a problem with my primers that covers my desired fragment because there is also smear at negative control!! I attach the gel photo.1st lane is F1-R3, 2nd is F1-R2, 3rd is F1-R3 neg., 4th is F1-R2 neg.
Can anybody say something and save my life please:) 
thank you...
Hi. I also have the problem of getting a band for the negative control. But it only happens when I use a specific set of primers. I have used the same template and same stock reagents and Taq with different primers and got clean negative controls, so there can't be contamination there. Also, I always use different lab coats, gloves etc, and master mix is prepared in a separate hood from where template is added. Could the primers be the problem? How can I solve it? I am very new to this technology so bear with me! 
Thanks.
Nevermind-
As you have no doubt read, anything in PCR is a possible source of contamination. If it is happening with only a specific primer set, I would suspect that it is one of the primers. Order new ones at the smallest scale from your favorite provider and see if it clears up the problem. Its far faste and cheaper than anything else you can do. 
katanin-
I've had similar problems on occassion. Are you using a hot start polymerase? This can sometimes solve the problem by preventing the primers from being extended until only the specific interaction is occuring. There are also kits available to help you get the reactions to work and are frequently worth the money because they save you weeks of work. The Failsafe system from Epicentre (http://www.epibio.com/item.asp?ID=294&CatID=7&SubCatID=102) has saved my butt on a couple of occassions. Despite what the companies say, getting PCRs to work over a kb can be quite challenging.
As you have no doubt read, anything in PCR is a possible source of contamination. If it is happening with only a specific primer set, I would suspect that it is one of the primers. Order new ones at the smallest scale from your favorite provider and see if it clears up the problem. Its far faste and cheaper than anything else you can do.
Thanks for the reply Scotty.
I have ordered and used new primers (twice from 2 different providers!), with the same result, sorry I didn't mention that in my first post! So that's why I was posting here, wondering if there was something I should know and don't, maybe about size of primers or whatever. But anyway, I'll try again! Lucky number 3, here I come!
Ok, it's all sorted out...changed everything again, with new primers from different company...and it's all good! Hooray!!!
Random contamination:
We've been having a ghost in our lab for some time now. We see random PCR contamination of wells, and haven't been able to figure out the cause.
If we run a 384 well plate of negative controls, a random number of them will result in false-positives for certain products in a multiplex. They don't always have the same false positive product, those seem rather random too. All the wells use the same master mix.
If all wells have the same master mix, what are the possible causes of the random contamination? Dust settling into wells?
How and where do you store your plates? Do you make your mix and add them to the plate in a laminar flow? Try a new plate, put it under UV for a long time, then add the master mix and perform PCR...
may be you should UV-irradiate even your pipettes...