mislabelled tubes?

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We had a severe problem with PCR contamination in our lab earlier in the year. One of my co-workers would run a plate several times, and always had a false positive. I would run the same plate with my reagents and, it would work fine. We irradiated everything in the hood, including pipettors, tips, gloves, trays, etc. I discovered one of the problems was that I would always irradiate my water, but our boss told her to NEVER irradiate water. The other was just her technique in general. We were looking for total absence of DNA in RNA samples. She would put her negative and positive control samples right next to each other on the plate, with the positive going into the plate first in A1. The she wouldn't cover the plate while adding the test samples. All of her samples would show slightly positive CT counts. When I set up a plate, I put my negative and positive controls in opposite corners and always put the positive in last.

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Thanks for your advise !

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hi every body

i have the same problem i have no amplified band after pcr carryover i repeat the pcr but i have the same result
all i have is primer dimer if the contamination is the same resone



can i put the water, buffer & dNTPs in uv 2 get rid of contamination or i will lose them
thx u very much

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There is one Carry-over prevention method available that can be used without much hurdle:

"Method for removal of contaminating PCR products from PCR or RT-PCR is subject to patent rights according to US patent 6,541,204 and equivalents. "
You will find more information at http://www.biotec.no/category.php/category.../?categoryID=82

It's easy:

1. Get the enzyme. It has many names: Shrimp nuclease, Shrimp DNase, dsDNAse, Heat-labile DNase,
2. Add one unit or so (depending on the volume of your mix) to your mastermix (=everything except your template)
3. incubate at about 37C for a few minutes
4. Heat inactivate at 70C for 10-15 min
5. Add template
6. Go on with your life

This is much like changing from CIAP to SAP when cloning; life gets easier!

The enzyme is available from GE Healthcare, USB, and ThermoFisher.

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if you do not need the PCR products for sequencing, cloning etc but just for restriction digests or some other applications such as real time PCR, one can use UNG (Uracil n glycosylase) and instead of using dTTP in the mastermix you can add uracil.
Then you start the PCR with an incubation step at 37 or 55 (depending on the UNG) for 15minutes followed by hot-start at 95 and the normal cycling.
If any PCR contaminants are present from previous PCR done using dUTP they will be digested during the initial step. The hot start at 95 will deactivate UNG and activates Taq polymerase. Genomic DNA is not digested because it contains T and not U.
UNG can be bought from Invitrogen.

good luck

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"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...)"

I have this same problem, and I can't figure it out. I have bands in my water controls, but no bands in some of my samples. How can this be? Everything that is in the water controls is also in the samples. The only difference is the sample dna is in the sample, and so the water has 2 more microliters of water relative to everything else in the tube. Anyone have any ideas about what could be going on?

Thank you very much,
Bok

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Hi everyone,

My RT-PCRs have become a trial and error game, I have been struggling with contamination in my negative NO template samples for the last many months.
I have read the reponses given here and tried most of them, i use gloves, filter tips, clean the bench with 95% EtOH, autoclaved the tubes and pipettes and am very carefull with pipetting, but i cnat get clean controls.

I have uploaded 2 pictures, a gel picture and a melt curve for the for you to see.

The gel picture:
Lanes 1-4 are samples with DNA template
Lanes 5&6 are negative control samples,
All the 6 samples were from the same master mix, same water, same primers, same sybr green mix. Why would i get contamination in one control and not the other.

Melt curve graph:

the control without contamination had a peak at a lower temp compared to the other samples (green graph, lane 6 on gel)

Am sooooooooo frustrated I dont know what to try next. Is it the lab, machine??

Please help..........

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I recommend NOT autoclaving your tips or tubes. Sterile is different from clean. Autoclaves do not remove DNA, but can add it. Have you ever looked at the inside of your autoclave?

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While it is true that using fewer cycles won't get rid of contamination the balance between the amplification of pos and neg is always relevant if using general bacterial primers. This is because the reagents always have a low level of contaminating DNA which is often bacterial so if you amplify the reaction for enough cycles you will eventually get a band to form in the neg control. Sometimes it takes more than 50 cycles which is about the maximum that the Taq will last so to get more than 50 cycles you have to do a nested set of PCRs but you will almost always eventually see a band in the negative PCR especially when using universal or general bacterial primers.

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