My pipettes have somehow been decontaminated with an amplicon and now I get false positives in all of my PCR reactions. Is there a way to decontaminate the pipettes without dismantling them because I think that would mess up the calibration on the pipettes?
Hi Rosa, here is a guideline but I guess you have to dismantling the pipette to make sure that they are safe again.
https://fscimage.fishersci.com/images/D16542~.pdf
All the best!
Cheers, Nadine
You can try to wash them with bleach and leave them under the UK for some time (overnight). It should be ok. The best is to have separate sets of pipettes for the different procedures (dna extraction, amplification ans post amplification procedures). I don't know if that is the case.
I agree with Maria, the best thing is to have different sets for different procedures, but in case you want to decontaminate the set that you already have you can try to clean the exterior with bleach, ethanol and RNAse Away to eliminate any enzymes that could degrade your nucleic acids and degrade any contaminations that you already have in the pipette. Also using filter pippette tips is useful in preventing these contaminations.
I guess Maria meant UV....not to keep under UK...Or else that would be even bigger problem than getting false positive...:-)..U can even autoclave your pipettes if it is allowed (some pipettes can be autoclaved)....Good luck..:-)
Hi Rosa, with all the above mentioned options, I would say- use filtered tips; this way you will not contaminate your PCR reaction with anything stuck in your pipette :)
To keep things simple, I always keep a spraying bottle filled with ethanol on my bench; every now and then I spray my pipettes, hands, bench surface etc with ethanol and wipe with a clean paper. I'm in good luck interms of having contamination in PCR rxns :)
I agree with the previous comments, UV, bleach + EtOH, and some commercial brands are also selling DNA decontamination solutions (i.e. DNAtor). I reckon though that the best way is to use filter tips.
Clean your pipettes with Takara DNA-OFF and then wipe clean with 70% ethanol, followed by 15-30 minutes of UV.
I suggest that you also use fresh aliquots of buffer, dNTPs, water and pcr tubes for subsequent PCRs.
I agree with Melissa Call. Follow the regular procedure of sterilization, even you can do autoclave provided the pipette should be of such type. Try DNA dilution buffer. Also there are chance to undergo miscalibration when pipette get contaminated. So clean, sterilize and check for calibration. If not call the technical person ask him to calibrate. Take standard pipette and your pipette. Set 5micro ltr to standard pipette and to set maximum measure your pipette. Using standard pipette pipette out buffer to a eppendorf. Now pipette the same buffer using your pipette. Don't pipette it out holding in tips gradually adjust it 5 micro ltr. Hope this could help you.
CHEERS,
Jesu
I use 10% bleach to clean my pipette, there's also a solution call trigene. use of filter tips would help.
Eppendorf, Gilson, they all provide pipette service, they can help you clean and calibrate the pipette and they have workshops ( usually free, with conditions) to train you on taking care of your pipettes.
Hope these help.
We generally use DNA zap Solution for cleaning pippetes and hood and then wipe them using 70% ethanol and treat them under UV light for 15-20 min. or You can just autoclave the autoclavable parts of pippetes.
Hi Rosa, a good possibility is that one of your primer stocks is contaminated; try with a fresh primer stock and see if you still get false positive in all rxns.
Hi Rosa, Some of this is repetition, but many excellent responses include some bad info.
1. Workflow: Try to keep work areas separate and work from low-template areas to higher ones (PCR reagents sample extractions amplification, agarose gels and cloning), and have dedicated pipettes, tips, and tube racks for each area. Also, make sure to change gloves between stations.
2. Do your controls: It’s more likely one of your solutions is contaminated, so start with fresh water, primers, etc., as well as a fresh box of tips. You can determine which one was contaminated after you’ve had good results
3. Use filter-tips, and wipe down your work area and pipettes with DNA-Off or something similar.
If your pipette is contaminated, you would have to remove the tip-holder part and soak it in DNA-Off.
Ethanol and autoclaving are useless at removing nucleic acid contamination. And UV light is also ineffective – it will not penetrate, and in most cases the lights will have outlived their effectiveness. Bleach (10%) is effective, but will eventually damage plastics and metals.
Good luck
I'm agree with Richard, but also you can include the use of UDG, not is the solution but you'll be able to follow your work
Hi Rosa, you can use DNAzap solutions from invitrogen. Please go to this link http://products.invitrogen.com/ivgn/product/AM9890 for further details.
Well Roza....Lots of good suggestions above....
If you have a spare set of pipettes, check your reagents once. if you still get false positive....think of a fresh set of reagents.
You can go for the maintenance of pipettes taking cues from company experts and over a period of time you will develop confidence. A proper dissembling and assembling will not interfere with the calibrations.
Daily use of 6-8 % bleach in addition to DNA Zap sprays will be handy...
Cheer up and go ahead..
Best wishes
MMM
Some micropipettes (eppendorf research...) could be autoclaved, you should try this way before chemical treatment.
Hi Rosa, try to moist a piece of tissue in 5% formic acid and wipe all parts of your pipette which are exposed to the aliquote. It will cause apurinisation of the contaminating DNA. After that do not forget to dry the pipette and re- calibrate it.
If you got contamination inside, you will have to use some sort of bleach/acidic solution or autoclaving after extensive rinsing with water or the above mentioned solutions. It will be hard to ensure full decontamination inside the pippet using UV. 70% EtOH will get rid of bug contamination but not nucleic acid.
Hi Rosa,
If your pipettes are contaminated, it's very likely that the contamination is on the inside of the barrel. In this case there is no alternative to dismantling the pipette - unless it is autoclavable. Take it to pieces, clean it with alchohol and/or hot water and detergent, soak it in bleach overnight, rinse, regrease and reassemble. It's a pain, but if you haven't got the money for a new set of pipettes, it's the only solution.
You may want to recalibrate the pipette, but I personally wouldn't bother going through the weighing method. Just pipette an appropriate multiple of the critical volume into a graduated tube. PCR reactions are robust, so if the pipette is a few percent off, it won't matter.
Note that using UDP will not help. The UDP-UNG system relies on incorporating uracil into the amplicons and then degrading them with uracil DNA glycosylase (UNG). If your contaminating amplicons were made without UDP the UNG enzyme won't touch them.
A lot of people still use ethanol for PCR decontamination. I can't understand why. DNA is insoluble and stable in ethanol - that's why we do ethanol precipitations. I think it must be a hangover from microbiology labs. Anyway, it doesn't work. Use bleach (which is cheap) or one of the proprietary DNA killers (which smell nicer). Acids ought to work too, though I've never tried them myself.
Filter tips should be used all the time for PCR work. They should help, but I wouldn't be happy setting up PCR reactions with just a filter between my reagents and a contaminated pipette.
Lastly, you should never, ever, use the same pipette for post-PCR and pre-PCR work. Most people use three sets - one set for primers and other reagents, one set for unamplified samples and one for post-PCR work. I colour-code mine with tape and threaten to do horrible things to anyone who takes them out of their designated area.
Good luck. My group contaminated a whole lab once and it took three of us 6 weeks to clean up. If you have an alternative PCR reaction that targets a different area, I'd order the primers now.
In such situation pipettes and instruments need to autoclave 20 min. 1 atm., but you must be sure that it can do for your pipettes ( look instruction or pasport of pipettes). You must do general cleaning work rooms (zone 1, zone 2): wash off walls, floor, lamin. box, than 30 min. UV. All equipments to wipe 70% ethanol. In conclusion for control of decotamination need to take smears all surfaces and to test it in PCR (detection amplicons). It's necessary for good results in future. Good luck!
Two easy solutions. Take the barrels off and soak in a 10% bleach solution. Then rinse thoroughly until no trace of Cl smell. Then put them in a 37 C oven overnight to dry out. Also, I used to have a cardboard box with pipette barrel shaped holes in the bottom. You can place this mouth-side down on a UV light box and let them get exposed for 15-20 min. But filtered tips are the best (but more expensive) answer.
Just a quick response to Richard Pilon's suggestion that UV is no good - I agree that sometimes UV disinfection cannot be great, however get a UV crosslinker - its about the size of a microwave and not too expensive, and you can put racks and tubes etc in them to completely decontaminate surfaces of small equipment.
It's not the cheapest or easiest solution - just an additional step you can take to minimise the chances of contamination events.
Always, working in a clean space is key however since you have contamination, it is necessary you dismantle your pipettes and clean with HCL solution. It happened to me once. All the best
Hi John,
Could you please elaborate on how you managed to get rid of your contamination?
Do I have to do a HCl decontamination followed by a putting the pipette parts in a base? I'm wanting to get rid of the contamination which doesn't seem to be on the surface. Also, were your calibrations messed up once you dismantled and reassembled your pipettes after decontamination?
Thanks so much!
I had once a very similar situation. It turned out that it was a contaminated reagent and I just was not able to accept it before facts. A very good advise that I got was to use someone else reagents (better from another lab) to compare results. If you get no false positives with another lab's reagents, I would suggest "do not waste time and get ride of all your reagents" (it sucks, I know, they are expensive, but more expensive is if you keep using them). If you really want to find the source, use a backup reagent at a time until you find the one that is contaminated. Before I accepted it, I had a good 10% bleach routine before and after using my pipets (ethanol does nothing to avoid contamination with nucleic acids) and overnight at 37 C, and once per week the UV crosslinker. Nevertheless, I would use filters and see if there is a difference with the results before everything else. Good luck.
I had that trouble once too and I was trying to decontaminate everything I use, my work space and even did my PCR in the hood. I guess you could try all the suggestions above but have you tried asking your colleagues to perform the PCR so you can eliminate the 'technical error' factor?
Hope this helps! Best of luck!
I use bleach and UV light to decontaminate. Even though expensive, filter tips are a must. When contamination comes in, it is not just a pipette but can be everything that has been used. Without proper precautions amplicons can easily transfer to "clean areas". It is also a good idea to have someone else run the test as that will let you know if technique plays a role in contamination.
Try to get some Autoclavables pipettes. For me, is the easiest and safer way to decontaminate them
Its good to use filter based tips if possible but if not possible than we can autoclave it as now every micropipette is autoclavable.
As far as i am concerned go for cheaper and best way.....so try above method.
I am doing this always but if it doesn't resolve problem means check the reagents once again for their purity otherwise this problem will be permanent for you
Best of Luck!!!!!
Try "RNase away" from Carl Roth GmbH + Co. KG
http://www.carlroth.de/media/_de-de/usage/2653.pdf
It should eliminate RNase but also DNA contaminations. In our Lab it works really well.
Much luck!
I agree that persistent contamination is more likely to come from a reagent - suspect everything, and doubly suspect any general reagent that may be shared across people working in the lab with you! Do use filter tips and dedicated pipettes and observe all pre and post PCR segregation steps to the letter.
Use two different lab areas for PCR setup and working with amplified DNA (even better two different rooms if possible). Use separate labcoats, gloves, pipettes, plasticware, centrifuges etc for your pre and post-PCR areas, and make sure nothing ever crosses between them. Clean all benches and pipettes regularly with 10% bleach, then soapy water. Although ethanol is great for sterilisation, it does not work to remove DNA - in fact it just dries contaminants on and makes them harder to remove.
Always centrifuge tubes before removing lids, pipette slowly and use filter tips - these precautions help to avoid the aerosols that are a major source of contamination. Change your gloves frequently.
You could spend a long time trying to figure out where the contamination is, as others have said its likely to be in your PCR reagents, etc. Its worth just getting rid of these and buying new reagents.
Try to expose your pipettes under UV light for several minutes before using them. Use filtered tips in order to ensure that any possible source of contaminants wont get though in your pipettes.
I agree with Juan Pablo. These products work. However, a spray bottle with ordinary household bleach will do the same job for a fraction of the price. That said, the proprietary products smell nicer.
dear all!
would anyone know some publications where concentration of bleach with time exposure are studied? which bleach are you using: tablettes dissolved in water? how to you clean pipets? it is not really possible to wet them completly in bleach? thank you for your help!
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