in your case enzyme may be the culprit
best wishes

I too am having severe PCR contamination issues. This has been going on for about 6 months.
We have gotten all new primers and reagents several times now and it is getting very strange and frustrating. We have cleaned all the pipettes and benches also changed the water.

I am the only person in the lab who has this issue and it is not a technique problem (i.e. hitting the pipette tip with dna on it accidentally into the negative reactions etc). I have to set up all of my reactions in a separate room in a hood, but no one else does. I have even set up a pcr that is totally a negative control, did not even take any dna out of the freezer and I had bands all over the place.

Also, we are working on two different genes and this issue only comes up when I work with the primers from one gene versus the other. Working in the hood helped for a few weeks and then yesterday I got a very strong positive band in my negative even when in the hood. I wear two pairs of gloves. I dont know what else to do, I am lucky to be working with wonderful patient people, but I am going crazy and feel like some kind of a freak. Someone else will set up a reaction and it will be fine and then I will set it up with the same reagents and it is contaminated. Any suggestions would be greatly appreciated.

Dear Friend,
i wish to suggest you something regarding PCR.
I have also faced similar kind of problem. in my case culprit was enzyme . some body has used pipette used for taking positive control plasmid for aliquoting the enzyme.

you must have a separate pipette used for positive control only. Just for positive control. because whenever you are using plasmid as a positive control there are more chances of aerosol contamination.
take care
best wishes

Dear Amit,

Thank you for your suggestion. However, all the pipette tips that I use have a filter on them to block aerosols from contaminating the actual pipette.

Dear Friend,
i wish to suggest you something regarding PCR.
I have also faced similar kind of problem. in my case culprit was enzyme . some body has used pipette used for taking positive control plasmid for aliquoting the enzyme.

you must have a separate pipette used for positive control only. Just for positive control. because whenever you are using plasmid as a positive control there are more chances of aerosol contamination.
take care
best wishes

QUOTE (elephantman @ Mar 11 2006, 08:40 AM)

I too am having severe PCR contamination issues. This has been going on for about 6 months.
We have gotten all new primers and reagents several times now and it is getting very strange and frustrating. We have cleaned all the pipettes and benches also changed the water.

I am the only person in the lab who has this issue and it is not a technique problem (i.e. hitting the pipette tip with dna on it accidentally into the negative reactions etc). I have to set up all of my reactions in a separate room in a hood, but no one else does. I have even set up a pcr that is totally a negative control, did not even take any dna out of the freezer and I had bands all over the place.

Also, we are working on two different genes and this issue only comes up when I work with the primers from one gene versus the other. Working in the hood helped for a few weeks and then yesterday I got a very strong positive band in my negative even when in the hood. I wear two pairs of gloves. I dont know what else to do, I am lucky to be working with wonderful patient people, but I am going crazy and feel like some kind of a freak. Someone else will set up a reaction and it will be fine and then I will set it up with the same reagents and it is contaminated. Any suggestions would be greatly appreciated.

I also hace severe PCR problem. I suspect it is contamination with plasmid DNA. I am trying to amplify cDNA, and I've been having strong positive bands in my negative control for weeks. I've tried a lot of different primer pairs, and I've bought new primers three or four times! I am the only person with this problem in the lab, and I think it is a contamination of plasmid with my cDNA cloned in (I did PCR with plasmid DNA time ago). I have no contamination problems with genomic primers.

I have worked in hood after UV-irradiating pipettes (a different set of another person), filter tips, water, tubes, buffer and magnesium, and with a brand new Taq. Contamination persists. Should I have to buy new primers again and again?
I need to have these PCR result soon!

while proceeding for cloning i screened for positive clones by colony pcr where only few colonies showed positive but my -ve control i.e. vector alone & water alone give bands corresponding to my insert. The confusion is if any of my reagent is contaminated then why all of my colonies are not showing positive in colony pcr?

Did you clean the filters of the hood!?!?
I have a friend who have two differentes chambers to prepare the PCR, one for the mix preparation and another only to pippet the template, and a exclusive room to this chambers, with only this two hoods and a refrigerator.
This room have positive air presure and UV everywere.

I'm having problems with random contamination on nested-PCR, this room for the PCR preparation is perfect, but I dont like the idea of bring the amplified product to this room to prepare the second pcr. I guess i'll need another room.

I work with PCR tubes, not plates, does anyone know if there is any diference on working with plates or tubes for the contamination issue.
Because, everytime I open a tube I can imagine a bunch of amplicons jumping from that tube to the others around, and since I work with diagnosis, there is always a lot o reactions running at the same time.

My masterthesis is about low DNA input in PCR -and i must admit that i'm amazed of how sensitive this method is

I had 6 months of trouble due to contamination and most of it was solved by all the above mentioned tips -and i cannot emphasize enough how important it is to have a separate lab with uv-light to set up mastermixes

But another sorce of contamination i have encounter is hot-start PCR or more specific - contamination is very easily achieved when the PCR tubes are opened after some time in the PCR machine. We have a relative new eppendorf machine, and if i was to stop pcr to add some additional reagents i almost always got a contamination in my negative control. My guess is that this is due to the vapour which occurs after heating.

oh and another thing : always change gloves... i believe one can never change gloves enough when setting up PCR

and i know how incredible frustrating contamination is, but don't give up, suddenly you will realize what the source is and then the fun begins

-good luck