10 replies to this topic

[Jessica Crowley]

Our lab is dealing with some very pesky contamination in our negative (water only) controls lately. It seems in some of our assays we are seeing the wildtype band, the transgenic band, or even both. The most confusing part is the inconsistency of it all. We can run three assays together, each with different primer sets and only one will show the contamination. That means the only thing changing is the primers, but we will have used the same primers in the past with no problems. Unless we have all suddenly forgotten how to do PCR, there is something fishy going on. Notes:

• dNTPs have worked fine for other Assays and QPCR
  
• We cleaned our pipets and always use barrier tips

Can anyone offer any advice? If we could at least pinpoint where it is coming from, that would help. Thanks!

[phage434]

Do you autoclave your water? Autoclaves are dirty.

[Jessica Crowley]

We do. However that would not explain why all of our samples are not showing at least the wt band.

[hobglobin]

The samples (and/or other solutions) are contaminated with wildtype DNA?

[Jessica Crowley]

Sometimes we see only wildtype, but sometimes both the wt and transgenic band. It usually shows up in the water negative controll and all the samples end up having the same genotype.

[mdfenko]

maybe your primers are contaminated?

[Jessica Crowley]

Right now we are to the point where when it deosn't work we just have to re-do it and hope for the best. Everytime it doesn't work we just replace the primers just in case, but I just don't think it's that. I honestly am starting to think it's our water aliquots. We just got these new colorful 1.5 ml tubes and they don't really close all the way. I think that is a link and I'm just going to blame the ungergrads for now too I think.

[Mimoza]

Have you cleaned your pipettes? Are you using filtered tips always? Maybe the contamination comes from the pipettes.

[jerryshelly1]

Sounds like you are isolating tail clips to determine whether your transgene is present?  Did someone double dip in the phenol or other solution for DNA preparation.  If you use NaOAc for precipitation, it is possible that you have contaminants floating bound to the salt that only get picked up a portion of the time.  I don't know.  Sounds fishy either way.

[Multiplexion]

Sounds like low level contaminations with PCR products in either any of the reagents, aerosols or materials you are using.
You do not detect them in every reaction since the concentration is too low.

[bigudukaz]

How close is the PCR preparation area and the gel area? If the room is not well ventilated and you ran the gel close to the PCR preparation - you may have some of the PCR fragments just flying around.

We had similar problem in our lab, and now we prepare PCR/qPCR in separate room, and runing gels in other lab (even changing lab coats) in 2 years we had only few cases of negative control giving a band.

Hope it helps somehow..