chronic PCR contamination - (Mar/10/2006 )
Hi
I had the same problem it turned out to be the taq, suppliers say it is due to some residual bacterial DNA in the taq! Even though my primers are metazoan 28s. Do a side by side PCR usig different Taq. to compare.
-sponge-
QUOTE (elephantman @ Mar 10 2006, 04:46 PM)
I am having severe PCR contamination issues that have 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. 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. 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 in my negative even when in the hood. I even 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 and it is contaminated. Any suggestions would be greatly appreciated.
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. 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. 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 in my negative even when in the hood. I even 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 and it is contaminated. Any suggestions would be greatly appreciated.
Your problems might be in pipettes, tubes, tips, hair, air, whatever.
You could try treating you mastermix with Shrimp nuclease, which degrades dsDNA.
Aliquot into several PCR tubes before incubating.
Then inactivate the enzyme and add:
template to one tube,
nothing (but put in the tip like you would if you added something) to another, and
do not open the third tube,
add clean water to one tube,
and so forth.
This would tell you where the contamination comes from, since the non-opened tube should be clean unless the contamination is comming after PCR which is quite unlikely.
-Gerd-