27 replies to this topic

[Martorse]

I am stuck with my PCR work for nearly 1 month due to contamination (false positives even when I set my reactions only with water). I already changed all stocks including primers, washed pipettes with bleach, I use filter-tips and wear gloves, perform pre and post-PCR operations away from where PCR is carried out. I even UV-irradiated the thermocycler for more than 3 hours. Can anyone help me?
Thank you in advance for the slightest hint.

[SVTX]

Don't forget the water!  Are you using disposable PCR tubes?

SVTX

[preeti]

while making mastermix for pcr try pipetting in all reagents along sides of pcr tubes rather than going down to the end of tube. later spin down all stuff on benchtop spin
also pipette slowly rather than just aspirating all reagents quickly

i have had worse situation than u

[thegradstudent]

PCR contaminations are like ghosts... you know they are there but you dont know WHERE??

I have followed the following with good results...

1. Prepare your master mix in a separate room from your current location, somewhere PCR is not the main practice... Be sure to have a separate lab coat, gloves, tubes, pipette tips tobe used only in that clean room.
2. Use a separate aliquot of DEPC water stock for each round of PCR
3. Prepare your mix in a hood with laminar flow. Decontaminate it with bleach, alcohol, RNAse, DNase, etc... Be sure to UV-irradiate pipettes, pipette tips, tubes, racks, gloves, and also your aliquots of water and PCR buffer... before the procedure.
4. Use a different pipette tip when pipetting all your reagents, even the same master mix to each tube
5. Keep your tubes closed during the procedure, even your master mix tube. Be sure that your tubes are closed when discarding the pipette tip!!! Aerosols are dangerous!!! Open the tubes only when necessary.
6. More important... schedule your PCR when not handling plasmids!!!

Hope this help...
Good luck

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[ocean]

it could be your gloves...an honors student in a lab near us had the same problem where she changed all her reagents and even used our buffers and dNTP's etc .  there was no contaminatin in our buffers or primers etc..eventually her supervisor found out it was her gloves..

if you make up your master mix first, change your gloves after you load your samples.  if you load your samples before you master mix then change your gloves just before you make up your master mix.

hope it heaps

[sharmamamta78]

is the band in the false positive very faint??? or is it similar in intensity
when compared with ur positive control?? If it is faint in case of false positive and very bright in case of positive control then you can also try reducing the number of cycles.
good luck!!!

[hula]

Quote

If it is faint in case of false positive and very bright in case of positive control then you can also try reducing the number of cycles.

Using fewer cycles won't get rid of contamination although it gives a false impression that there is no contamination.

[korsh ararat liandamahar]

SVTX, on Oct 1 2004, 01:42 PM, said:

Don't forget the water!  Are you using disposable PCR tubes?

SVTX

    thanks for your work

[Martorse]

Thank you all, apparently contamination disappeared as it came. I only wish it never ever happens again. I keep UV-radiating every stock except primers and taq and follow each of your tips. Thank you again for support...

[Martine]

Hi all,

I also have problems with qRT-PCR contamination in the NTC's.

I think I really tried everything: washed my pipets and UV radiated them, also tried pipets from someone else, changed my reagents (the water, the SYBR green Master Mix, ordered new primers and even new primers with another sequence in totally different exons). There is always contamination for my housekeeping gene but not for my gene of interest (There my NTC's are always nicely 40, so it would in my opinion not be a problem of the water, the Master Mix or the pipets I use)... The Ct-values for my housekeeping gene samples are between 16 and 23, but the Ct-values are very close to that (24-25) so that's to close to trust the Ct-values of my samples.
When I put the NTC's of my housekeeping gene on gel, they give the same band (also the same visibility) as my housekeeping gene in a sample with mRNA, so it's not a problem of primer-dimers.

Any suggestions?
Thanks in advance
Martine

[Martine]

Hi all,

I also have problems with qRT-PCR contamination in the NTC's.

I think I really tried everything: washed my pipets and UV radiated them, also tried pipets from someone else, changed my reagents (the water, the SYBR green Master Mix, ordered new primers and even new primers with another sequence in totally different exons). There is always contamination for my housekeeping gene but not for my gene of interest (There my NTC's are always nicely 40, so it would in my opinion not be a problem of the water, the Master Mix or the pipets I use)... The Ct-values for my housekeeping gene samples are between 16 and 23, but the Ct-values are very close to that (24-25) so that's to close to trust the Ct-values of my samples.
When I put the NTC's of my housekeeping gene on gel, they give the same band (also the same visibility) as my housekeeping gene in a sample with mRNA, so it's not a problem of primer-dimers.

Any suggestions?
Thanks in advance
Martine

[phage434]

Use tips (filter) and tubes directly from the (new) package, not autoclaved or handled in any way.

[Sonia]

I'm also encountering the contamination problem. I run 2-step RT-PCR, the NTC also give product whose intensity is as high as the samples. I already change the new reagent and prepare the master mix in the preparation room. Is it possible that the aerosol give such heavy contamination?

[terrycui]

Sonia, on Jun 1 2009, 08:23 PM, said:

I'm also encountering the contamination problem. I run 2-step RT-PCR, the NTC also give product whose intensity is as high as the samples. I already change the new reagent and prepare the master mix in the preparation room. Is it possible that the aerosol give such heavy contamination?

strong false-positive intensity, but not a weak band, from aerosol contamination makes no sense (unless ur lab is dirty enough)

1. double-check all of ur reagents (water is the most one u should consider with)
2. take care with ur hand with which u open the tube ( it is possible that DNA remain on ur hand and brought into other tube which makes contamination)
3. take care with ur tip, never ever touch unexpected place

good luck

[lavoij]

I am also having some issue with, contaminations but it is extremely bizarre, it is not in all wells! When I repeat my PCR I do not always get the same positives (this a PCR for genotyping) and sometimes I get a positive band in my blank. I have tried changing all my reagents (including my water), cleaning my pipettes, even using somebody else's pipettes with their tips. I am not sure what to do anymore. I do not have many choices for my primer sets as the transgene is very similar to the endogenous gene. Any suggestions?

Edited by lavoij, 15 June 2009 - 09:13 AM.