I know of several cases in which random contamination appeared due to the people working just on the receiving end of an air conditioning vent. The almost non-noticeable wind current can make micro-aerosols from your samples fly off the tubes or pipet tips and fall into your blank tubes. I know it sounds strange, but in the cases I mention, the contamination problems finished just by turning off the air conditioning while preparing the PCR experiments!

Our lab has experienced many contamination problems in the past 2 years while using fungal endophyte markers for PCR. We, too, went through all of the precautions of using a flow hood, UVing surfaces, using Master Mix, new reagents, etc., etc., etc. I started hunting in the literature and found a paper explaining how common airborne fungal contamination and fungal-contaminated PCR reagents from the supply company are. Here is the citation:

Loeffler J, et al. 1999. Contaminations Occuring in Fungal PCR Assays. Journal of Clinical Microbiology 37(4): 1200-1202.

It may be useful. Also, cloning the false-positives and sequencing them might tell you whether or not it's fungal and how to proceed. If it's fungal, it will be much more difficult to deal with then if it is carryover contamintation or something similar.

Hi all,

I have recently used Clonetech's Universal genome walker kit with pretty good results. I did have to do a bit of tweeking though.

First of all, the annealing Temps. suggested in the kit didn't work, not on the controls or my specific samples. I went to gradient PCR to figure out the best temp for each combination of primers (adapter & specific). Next, after doing all of this trouble-shooting, I was out of Taq and didn't want to spend an enormous amount of money to replace their proprietary Taq, so I tested a few and found that Invitrogen's Platinum Hi Fidelity Taq worked well at about 1/6 the cost of their Taq.

As for cloning vs. direct sequencing, I never direct sequence unless I'm > 90% sure I'm only amplifying one product. When walking through a 'library', there is a high chance that you will get more than one product in any PCR reaction or gel purified band for that matter. I use pGEM-T easy, love it, have cloned larger inserts (2.5 kb from this walk) and it's easy to sequence using standard primers.

My organism is ryegrass and I've obtained additional 5' and 3' sequences, but still not to the ends of the gene. The trouble is that there are introns in my genomic DNA, so now I'll resort to RACE!

ooops..cut and pasted to the wrong topic. Sorry!

Hi all,

I have recently used Clonetech's Universal genome walker kit with pretty good results.  I did have to do a bit of tweeking though. 

First of all, the annealing Temps. suggested in the kit didn't work, not on the controls or my specific samples.  I went to gradient PCR to figure out the best temp for each combination of primers (adapter & specific).  Next, after doing all of this trouble-shooting, I was out of Taq and didn't want to spend an enormous amount of money to replace their proprietary Taq, so I tested a few and found that Invitrogen's Platinum Hi Fidelity Taq worked well at about 1/6 the cost of their Taq. 

As for cloning vs. direct sequencing, I never direct sequence unless I'm > 90% sure I'm only amplifying one product.  When walking through a 'library', there is a high chance that you will get more than one product in any PCR reaction or gel purified band for that matter.  I use pGEM-T easy, love it, have cloned larger inserts (2.5 kb from this walk) and it's easy to sequence using standard primers.

My organism is ryegrass and I've obtained additional 5' and 3' sequences, but still not to the ends of the gene.  The trouble is that there are introns in my genomic DNA, so now I'll resort to RACE!

I've been stuck with my pcr for almost 6 months. I'm getting the correct size bands in my negative RT-pcr, which is without superscript. I've tried everything testing for contamination, and nothing is contaminated, but I still am getting the correct bands in most of my negative controls. Does anyone know what else I should do, because I've tried everything and have failed miserably. Any suggestion, besides contamination, would greatly help. Thanks
ghezal

Hi ghezal,

One possibility outside what everyone has mentioned here in terms of contamination is that you are PCR amplifying a region within a single exon. Are you DNAse treating your RNA preparations "effectively" before doing your RT-PCR if that is not efficient you will still have genomic DNA in the negative control which may act as template. You would get the same length band if your PCR fragment is within an exon.

A second possibility could be if the band is small molecular weight it could be caused by primer dimer addition of DMSO or BSA in the reaction should get rid of it if that is the cause.

Hope this helps

Scott

For particularly difficult contamination problems I've read that 8-MOP is a useful agent to destroy DNA's ability a act as a template for PCR. Practically the procedure involves making the master mix with 25ug/mL 8-methoxypsoralen, then exposing the entire mix to UVA for a few minutes (just through the side of the tube at a distance of a few cms) before aliquoting to individual tubes and adding template. Interference with polymerases is apparently minimal.

If all else fails this may be an option; a reference in which it has been reported to have helped with a particularly sensitive protocol is Biologicals (2004) 32:183-193.

Hope that's useful for the really sensitive protocols,

Mark

Hi, Omar,
I have struggled for the contamination of -ve control for a month. It's terrible. I suggest you refresh all the reagent you used. As what others mentioned, water is one of the main source of contamination. But sometimes it maybe a carry-over contamination in your reagents. I do think that it will waste time to check which reagents cause contamination (I have tried it, even though you find it out, what you can do is to get a new one from the stock or even order another one). Hope that it will help.

Good luck

So far we've found that our main source of PCR contamination is the people themselves. If a person handles amplified PCR product, that product gets all over them, even if there is extreme care taken. I believe it is just the aerosol of the PCR product that gets on the face, hair, etc.

If the person then goes on to prepare subsequent PCR reactions, there is a high liklihood that some of the wells will be contaminated with previous PCR products.

The workaround we've found to this is to simply set up all pre-PCR steps before handling any post-PCR product.

It seems that a shower and a clean set of clothes is enough to prevent the contamination problem. Of course, this is only practical on a daily basis.

And yes, we do have different sets of lab coats.