Hi,

I'm also having problems with contamination of my negative controls. I'm working with 16s rRNA genes trying to amplify them from genomic DNA from acidophilic bacteria for identification.

I've tried switching out all of my reagents, switching out my water, switching out my mineral oil (and even autoclaving it), re-autoclaving all of my materials, washing everything down with a 0.1M HCl solution, switching gloves between every step, turning off fans (but not the normal circulation in the room, we don't have a switch for that), etc, etc.
I'm just wondering if anyone has any suggestions and if anyone has had any success in using a laminar flow hood for all their work?

I'm thinking it's possible that given that its springtime I might be getting fungal/bacterial contamination from the air and the cells are becoming lysed and amplified in my reactions.

Oh, also, the band I'm seeing is at the same spot as my desired band for the gene and its the same intensity when visualized with ethidium bromide.
Any help/suggestions would be much appreciated. It's been 6 months or so and I'm insanely frustrated with this.

lildoc82

Hi,

I'm also facing contamination issue for almost 1.5mths.... I've changed reagents, re-ordered primers for 3x, cleaned my whole bench with detergent, changed my sitting location, cleaned my pipettes and even asked another person to carry out the same expt, but still couldn't solve the problem.

The strange thing is only 2 out of my 5 sets of primers were 'contaminated' and when I tested the individual primer (take 1 pair of uncontaminated primers, mixed with either the forward or the reverse of the 'contaminated' primers), there seem to be no contamination. But when I mixed the forward and reverse of the 'contaminated' primers, a bright band in my negative appear (even brighter than my DNA sample) and its the same size (~100bp) as my expected dna pdt.

I would appreciate it very much if anyone could give me any suggestions....

Another question is can primers that form hair-pin loop be self-amplified?

Thank you!!!

Another question is can primers that form hair-pin loop be self-amplified?

Definitely. The critical factor is whether there is a 5' overhang in the folded hairpin. If so, then the PCR will very gladly add bases to the 3' end of the primer, making it not match your desired template.

I'd suggest that this may not be contamination, but problems with primer design.

There can be other intractable issues. You might try avoiding autoclaving anything coming near your PCR reactions (tubes, water, buffers, tips, etc.). There is a lot of confusion between "sterile" and "clean." Sterile does not mean lacking in DNA. You can have highly contaminated, sterile solutions. Try using tips, tubes, reagents etc. which have never seen a tube or bottle which has been near your autoclave. Barrier tips help. There can also be DNA contamination of some reagents, from some suppliers, such as Taq, particularly with E. coli DNA.

Just wanted to let everyone know that I found the problem. I called the manufacturer of my Taq polymerase that I was using and they were having problems with certain lots being contaminated with dna. I've since switched to a different taq and am seeing clear blanks once again!
Thanks everyone for the input.

lildoc82

What brand of Taq do you use? Maybe others are having this problem too?

I believe I had the same problem in my Hot Start PCR.... in which I add the taq while the tubes are still inside the heat block... the A/C in the room is newly fixed and there is a gush of air all the time ... well it keeps the room and the mechines cool.... having thrown away all the reagents except taq, I tried adding taq into the mix outside the room then the problem was solved....

Hello

I'm too, stucked with contaminations for some time now... and I need these results to finish my thesis :-(
Recently, I had two contaminations (appart from all the other ones before that) for which I can not find any answer :
1) I amplified a product cloned inside a plasmid. This segment is seabird DNA, I'm the only one to work with in the lab. I cloned in different plasmids fragments of different size. After this PCR, I have a very strong result in my negative (my negative containing only PCR mix), and strong bands of the expected lengths for each other plasmid. I would have expected at least to find the same "contaminating band" (corresponding to the most widespread band in my plasmids), in every other sample...
2) I did a PCR on genomic DNA, on individuals that worked usually only half of the time... and again I found a strong band in my negative, but for some of my PCR, I had nothing... if I contaminated my reagents, I would expect to find the same band in every PCR tube...
(these two pcr were done using the same reagents, I will try again with new ones... but I can't understand that the contamination I saw come from the reagents, since I do not have the same band everywhere...)

If you have any explanation, that might help me to get rid of this awful - unpredictable - contamination..
Thanks

Liteul RV