I keep getting contamination in my negative control PCR samples. When i worked in other labs people were less careful of contamination but nothing ever happened, but now I'vw been very careful already but it comes back once every 2 months.
any suggestions??

There are so many ways to contaminate your PCR reaction it is basically impossible to help you. My only suggesting is to make small aliquots of all your reagents and throw them out when you start getting contamination.

Daniel

would you kindly post a photo of your gel?

sure, Its in the last lane, I loaded 15ul of a 20ul PCR product, i'm not sure if u can see it, its only very faint, but it was clearer on the geldoc.
The other gel was after cleaning my pipettes but using the same solution, ignor the top half of the gel, but you can see a few bands in the negative controls in the right hand side of the bottom gel.
thanks
flo

Do you use filter tip when aliquoting your samples?

The 'contaminating' bands look like your experimental target bands. I think you are getting template carry over. Filter tips may help. Are you running rxns in a plate. You could have well to well crossover contamination. Try using tubes or strips when setting up. Use caps or mats to cover in the thermalcycler. Don't use adhesive, foil or seals. If you still have a problem its in the buffers.

I'm seeing something that looks like primer-dimer too, but the gel is difficult to make out. I am referring to bands running smaller than the buffer; I am guessing the bands above the buffer are your target? and the two that are visible in the lower right of the second gel are the contamination that you are worrying about in your neg control?

anyways, I agree with everything said above

also, are you separating out your work areas, too? if this contamination shows up after a few months, then you may have aerosolized template crawling into your tubes too.

Sorry, can I just ask:How can the templates get aerosolized?? because the lab temerature is quite cool.
thanks!

yeah they are primer-dimers... have you got any tips in getting rid of them?
because sometimes they are there and sometimes they are not, even when I've being doing everything the same (I think)
thanks!