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-   -   Positive results for negative control runs!!! (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/74246-positive-results-negative-control-runs.html)

Alicialsw 11-08-2010 09:03 AM

Positive results for negative control runs!!!
 
Argh!!! I'm having a pretty bad week as I've been getting bands on my negative controls. I've done negative controls without template, without primers and without dNTP. I've even change the water every single time. The problem I'm facing is, I'm getting the same exact size band with my expected size and to make things worse, the band is even brighter!!! I've tried using my colleagues pipette, water, PCR reagents and rediluted my primers from the stock primer solution but nothing seems to be working. I kept on getting bands on my negative control (minus template) which is the exact same size with my expected band (which I sequence and is the gene of interest).


Please help me out here. I'm trying my very best to troubleshoot but I have no idea what is the contaminant. Sigh......

danfive 11-08-2010 04:23 PM

Re: Positive results for negative control runs!!!
 
Quote:

Originally Posted by Alicialsw (Post 423697)
Argh!!! I'm having a pretty bad week as I've been getting bands on my negative controls. I've done negative controls without template, without primers and without dNTP. I've even change the water every single time. The problem I'm facing is, I'm getting the same exact size band with my expected size and to make things worse, the band is even brighter!!! I've tried using my colleagues pipette, water, PCR reagents and rediluted my primers from the stock primer solution but nothing seems to be working. I kept on getting bands on my negative control (minus template) which is the exact same size with my expected band (which I sequence and is the gene of interest).


Please help me out here. I'm trying my very best to troubleshoot but I have no idea what is the contaminant. Sigh......

There must be alot of contamination floating around if no dNTPs/no Primer neg controls still show a band. Meaning primers and dNTPs are also available from whatever contaminant source.

1st. Are you new to doing PCR? If so you need to work on your technique, etc etc. [Don't want to bore you/discourage you by harping on this; only you will know if it applies to you]

2nd. You need to clean up, wipe down your work area, and any area nearby. Use Ethanol/Isopropanol or Bleach.

3rd. Toss reagents. Get new reagents (even stock primers), autoclave water, tubes, tips, the whole works. Take your pipettors apart (carefully) and decontaminate them (ethanol or 10% bleach). Use UV lights for decontamination at the end of every single day, if available.

4th. Separate the extraction, PCR setup and PCR machine and gel electrophoresis equipment out. Run them in separate parts of the lab or even rooms if available. (this is to cut down on aerosol contamination).

5th. Use gloves and change them frequently.

6th. Don't store reagents in same fridge/freezer as any pcr amplicons/gDNA. Keep them separate to avoid contamination, don't want them in the air when you open a reagent or PCR rxn tube, or landing on surfaces that will be touched and the contamination carried into new PCR rxns.

danfive 11-08-2010 04:32 PM

Re: Positive results for negative control runs!!!
 
Quote:

Originally Posted by Alicialsw (Post 423697)
Argh!!! I'm having a pretty bad week as I've been getting bands on my negative controls.

Please help me out here. I'm trying my very best to troubleshoot but I have no idea what is the contaminant. Sigh......

Suspect that the contaminant is PCR product/amplicon. Treat your finished PCR rxns very carefully to avoid sending the amplicon into the air. Kinda treat those little tubes like they hold toxic waste.

Next due to the info you mentioned. I would suspect that maybe a lab coat, lab book; something on your person could be carrying some of the contaminating amplicon. So please change those things out.

If you have a PCR hood, this could be holding the contamination, then irradiating with UV light for 60-90 minutes at the end of the day will "kill" contaminating amplicon in there. You can also wipe it down before and after you use it with 70% Ethanol.

Alicialsw 11-10-2010 06:31 PM

Re: Positive results for negative control runs!!!
 
Thanks for both of your suggestions. Nope, I've been doing PCR for like months already and positive results were pretty much well, correct.

I've tried most of the minor cleanup stuffs like cleaning the work bench, pipettes, changing the reagents (I even got my colleagues to spare me some of their PCR reagents, water and pipette) and I just ordered a new set of primers.

I'm suspecting that the original primer stock was already contaminated with the amplicon/template as the negative control without the template showed positive result (I re-diluted the primers) while the negative control without primers didn't show any bands.

If the band is still there, I think I'll have to extract new RNAs and do the DNAse treatment once again. I wish we had a PCR hood and I hate spraying liters of 70% EtOH and DNAse away all over the place.

Keeping my fingers crossed that the new primers works out fine. Have a good day and thanks :)

forum123 03-14-2011 09:23 AM

Re: Positive results for negative control runs!!!
 
hey all, I have the exact same problem...

is a PCR hood really that important? I do not have it... I do not know where I contaminate my stuffs but is PCR really that sensitive that some DNA in the air will cause it to amplify the same thing?

any tips to help me reduce contamination... I feel bad for contaminating my collegue's mastermix... please help!

danfive 03-14-2011 05:15 PM

Re: Positive results for negative control runs!!!
 
Quote:

Originally Posted by forum123 (Post 426616)
hey all, I have the exact same problem...

is a PCR hood really that important? I do not have it... I do not know where I contaminate my stuffs but is PCR really that sensitive that some DNA in the air will cause it to amplify the same thing?

any tips to help me reduce contamination... I feel bad for contaminating my collegue's mastermix... please help!

If a reagent is contaminated you should toss it and order new reagents.

Same thing with buffers/bottles that can be dumped, autoclaved, made from scratch.

You should decontaminate surfaces and pipettors with 70% EtOH on a regular basis. If you can safely autoclave equipment, racks, tip boxes etc then do so.

Track down the contamination by setting up reactions with only one suspect reagent (variable) at a time.
Like testing used PCR buffer vs New PCR buffer--only difference is the buffer all other reagents exactly the same.

From the above scenario substitute:
PCR buffer --Taq--Primers--dNTPs--H20--MgCl2
(use filter tips, change to non-suspect pipettors, change gloves often, keep tubes closed as much as possible)

forum123 03-16-2011 04:42 AM

Re: Positive results for negative control runs!!!
 
thanks for the reply.. i will try again... wasted all my reagents n efforts...


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