PCR contamination - (Oct/09/2007 )
Hello everyone!
I'm new to this, so any mistakes hopefully can be forgiven?...
Here is my problem.
Working in a diagnostic molecular biology lab, running various applications and never had a contamination problem before, if that is possible! I've been facing a persistent contamination problem in one of the applications, which involves HPV genotyping. It seems that a certain HPV pattern keeps contaminating almost all the patient, positive and negative control samples (I write almost, cause in 3 out of 20 cases the patient samples weren't contaminated with the pattern, although the negative control wasn't clear).
I use:
-disposable filter tips (apart from the P10 tips, which are not filter ones),
-UV irradiation twice a day, before and after PCR for at least 15 minutes (I even leave it on overnight sometimes)
-disposable tubes, which I autoclave before use
-ultra pure water, which I also autoclave before use (in aliquots)
-70% EtOH and 10% bleach in order to clean all surfaces
-2 sets of pipettes, one set used ONLY in the laminar flow which is brand new and used ONLY by me
-3 different working rooms, one for DNA isolation, one for PCR mastermix preparation and one for post-PCR applications (the 2 first rooms are UV irradiated daily)
I have thrown away all reagents, started using new ones, have bleached all pipetes thermocycler wells (!) and still the contamination persists.
Any advice or hint is more than welcome, since I'm about to lose my sleep over this....
is it possible that your hood is contaminated? Tissue culture hoods, for example, recycle their air whereas Chemical Hoods have a draw down. Otherwise... maybe you should try designing some new primers? Sounds like a tough one...
[quote name='jonathanjacobs' date='Oct 9 2007, 07:51 PM' post='113727']
is it possible that your hood is contaminated? Tissue culture hoods, for example, recycle their air whereas Chemical Hoods have a draw down. Otherwise... maybe you should try designing some new primers? Sounds like a tough one...
[/quote
The hood is a class II cabinet with new filters, so I don't think it is the proplem. Besides, all other applications are contamination free and I only use the hood for PCR mastermix preparation-the DNA sample is added to the reaction later, in another room.
I use a commercially available kit for HPV genotyping, which is CE IVD, and the primer mix cannot and should be redesigned...
your autoclave may be contaminated.
what was different about the three tubes that did not show the artifact?
maybe a kit component is contaminated.
what was different about the three tubes that did not show the artifact?
maybe a kit component is contaminated.
Actually, nothing what so ever! These 3 tubes came from 3 different PCR batches. These PCR batches had more than one samples, all contaminated apart from that one sample! In each batch of reactions I used a different batch of reagents (I discarded the reagents each time!!). Different batch of autoclaved tubes, etc. I have changed all kit components more than 3 times, even the polymerase, dNTPs, MgCl2 and 10x buffer, all of which are not included in the kit!!!
I suggest you stop autoclaving your tubes. It causes many more problems than it solves. Use the tubes from the box. And the water from your milliQ machine without autoclaving it (or buy pure water).
the autoclave should be cleaned out once a week. If it is the autoclave (as most here seem to suspect, and not any of your other equipment -tubes and pipette, reagents), try bleaching it. Then washing it out with lots of water. And see if the problem goes away.
Hello, I'm back from work.
Thank you all for your suggestions!
Unfortunately, it is not the autoclave. I conducted PCR with Ultra Pure water out of a new bottle, and new tubes, not autoclaved. I also used a new primer mix, from an amplification kit of different lot number than the other ones...I think I'll try again with nuclease free water from another supplier, but I can't use other Taq, dNTPs, MgCl2 or 10x buffer, since I'm the onlyone in the building conducting PCR. I only wish I could think of another possible source, since the contamination took place while I was on vacation and my co-worker claims to have done everything by the book....If it is the pipettes, what can I do to really clean them?
However, I'm positive that the contamination occures during PCR and not gel electrophoresis or reverse hybridization, since I used plain water instead of PCR product on a strip and at the end of the examination it came out clean. On the contrary, the real negative control gave the same contaminant pattern on another strip (same hybridization batch as the plain water sample). The odd thing is that in order to see the contaminant band on an agarose gel, I have to electrophorise at least 20ul of PCR product, while I only use 10ul for reverse hybridization).
could the contamination be coming from the template itself? Soundbody might have contaminated the sample DNA either while working with it or while making it.
Do you have any DNA of the same kind but you are certain is clean.
Do you have any DNA of the same kind but you are certain is clean.
Well, I have used a new positive control, which is supplied along with the amplification kit and is of known pattern (it is an already isolated DNA, that generates HPV type 6). I am the first to have used it and that came out contaminated as well (I can only use it for applications downstream DNA isolation). Then, I used a clean DNA isolation negative control sample (I use the QIAamp DNA Blood mini kit for isolation) from an application different than HPV genotyping (one that came from a cystic fibrosis application) and when used it for HPV PCR, it was contaminated!