PCR negative control contaminated and not the other wells - (Oct/12/2008 )
Hi,
I am doing a PCR to screen colonies that I have just transformed and I get a contamination band in the negative control which has exactly the same size as my target band, around 251bp.
But the problem is that I got this contamination band in the negative control, but not in all of the wells. On the 11 screend colonies, only 5 have the target band.
So it is really weird, because if I have a contamination, it should be in every wells. All the colonies tested should be contaminated!
I really need help!
Thank you very much
xavier
I am doing a PCR to screen colonies that I have just transformed and I get a contamination band in the negative control which has exactly the same size as my target band, around 251bp.
But the problem is that I got this contamination band in the negative control, but not in all of the wells. On the 11 screend colonies, only 5 have the target band.
So it is really weird, because if I have a contamination, it should be in every wells. All the colonies tested should be contaminated!
I really need help!
Thank you very much
xavier
unfortunately, there is no defenitive answer for your question as far as I can think of
that could be you just mixed up tube, or tube/pipette tip for the negative control was contaminated and so on
but either way, what you can do is repeat the PCR one more time, to see you will get similar result or not
one thing that you can say is at least water and PCR reagents that you use was not contaminated.
I am doing a PCR to screen colonies that I have just transformed and I get a contamination band in the negative control which has exactly the same size as my target band, around 251bp.
But the problem is that I got this contamination band in the negative control, but not in all of the wells. On the 11 screend colonies, only 5 have the target band.
So it is really weird, because if I have a contamination, it should be in every wells. All the colonies tested should be contaminated!
I really need help!
Thank you very much
xavier
unfortunately, there is no defenitive answer for your question as far as I can think of
that could be you just mixed up tube, or tube/pipette tip for the negative control was contaminated and so on
but either way, what you can do is repeat the PCR one more time, to see you will get similar result or not
one thing that you can say is at least water and PCR reagents that you use was not contaminated.
something that happened to me once was that a drop of one sample got into my negative control. We are using 8-strip-tubes for PCR that have to be closed by 8-strip-lids (which are separate from the tube). When opening the tubes after PCR the whole lid-strip has to be removed from all 8 tubes and depending how the lid-strip is removed, liquid can snip from one tube into the other....so I contaminated my negative control.
well, i use single tubes.
The second time I have repeated the PCR, I have been very carreful. I have done the PCR with only negative controls. And I have the same band! the same contamination!
But howcome some results could really be negative if even the negative is positive?
Kind of funny! except I lost my week end!
that's some weird stuff, assume you wasn't drinking night before the experiment 
, i suggest to move on w/ your experiment since you got the band you are looking for right?
wolverena: i once used that kind of pcr tubes and decide never to use them again after spilled like half of the sample from one tube into another tube during the opening process