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PCR: The Right Way to Decontaminate and Eliminate False Positives

PCR is highly sensitive, but the downside of that very property is that it makes the technique prone to producing false-positives. In labs where PCR is a staple, like the one I work in, any false-positives are more often than not due to amplicon contamination.

A broken capillary or a PCR plate left carelessly at the table edge is all it takes to aerosolize those amplicons. The next thing you know is it shows up in every PCR reaction that you run.  If you frequently run PCR, then you know what I am talking about.

So what do you do to decontaminate?

Well a simple, but effective way to combat an amplicon contamageddon, is to wipe down everything, equipment, workstations and pipettes, with bleach. Yes, bleach… not HCl. If you routinely use HCl for decontaminating rogue DNA then you definitely need to read on.

Sodium hypochlorite, the active ingredient in bleach, was shown in a 1992 study to effectively protect against amplicon contamination by causing extensive nicking, which prevented the 600bp fragment they tested from being amplified by PCR (see A. M. Prince, L. Andrus PCR: Biotechniques 1992 Mar;12(3):358-60).

The same study showed that even a after 5 minute exposure to 2N HCl, that same 600bp fragment could be detected by PCR. So HCl is not up to the job… its time to get the bleach out.

Which bleach should you use?

The efficacy of bleach in DNA decontamination (and as a disinfectant) depends on the amount of free and available chlorine. 0.05 – 0.5% of free and available chlorine is considered an intermediate level disinfectant and a commercial bleach (like Clorox) contains 5.84% available chlorine so a 10-100x dilution from a commercial stock will work just fine.

I tend to use a 10 dilution of Clorox and, in the above-mentioned study, Prince and Andrus used around a 20x dilution. You can make your own choice.

How and when to decontaminate

Generously spray workstations/equipment/pipettes with 10% bleach, then let it sit for 15-30 minutes (coffee break time!). Then wipe up the bleach and follow up with a water rinse and wipe — bleach is corrosive, so it will damage materials if residue is not removed by rinsing with water.

This procedure should be carried our before and after each PCR, and definitely after a spill. I religiously stick to a weekly cleaning routine of all of my PCR equipment and workspace and that has gone a long way to preventing amplicon contamination

Important points about diluting and storing bleach

Use clean water to dilute the bleach

I use regular tap water and that has worked well so far but hypochlorites are very unstable and decompose very quickly with hard water, reducing the availability of chlorine. So you may want to use a purer water, just to make sure.

Make fresh dilutions as often as possible

Potency of bleach will reduce over time so, it is essential to regularly make fresh dilutions. If you do not smell the chlorine in the bleach, then it is time to make a fresh dilution. We keep our (1:10) dilutions for about 1-2 weeks.

Store dilutions at room temperature in opaque containers

We use regular plastic spray bottles for the dilutions and store them under the sink. Temperature, light and oxygen are all catalysts for decomposition of bleach.

Make sure you use the correct PPE when handling bleach

Lab coat, gloves and safety glasses…. But you do that anyway, right?

11 Comments

  1. Kelly on April 4, 2019 at 1:56 pm

    The issue I am trying to resolve is the water to rinse with. Even if you buy RNase/DNase-free water to use as a rinse aide, the moment you open it, it is no longer sterile. You wouldn’t be able to autoclave as autoclaving doesn’t destroy all DNA. Does anyone have any thoughts on how they prevent water contamination? Or the verbiage they use in their procedures that allow non-sterile water to be used as a rinse aide?

  2. Maddy on February 23, 2019 at 12:03 am

    Hi Shoba,

    Thanks for the very useful article. Where did you read that a 15-30 minutes soak was necessary?

    Thank you
    Mad

  3. Rachel on August 15, 2018 at 4:01 pm

    I’m a bit worried about the health effects of decontaminting with 10% bleach once a week. It’s not particularly nice stuff, I find my lungs get irritated and my eyes sting. Would it still be effective to use a lower concentration and to decontaminate less frequently?

  4. sonia de rose on February 1, 2018 at 11:35 am

    Dear Shoba,
    thanks for your heads-up. We have a huge problem of contamination in my lab. we have already sprayed and cleaned all the surfaces with bleach (i think even more concentrated then 1:10); yet when we try to run a PCR to check the state of our hoods, it results positive even though the semples we run should be negative. the neg ctrl stays neg though. what do you suggest?

    Thank you,
    Sonia

  5. Wei Yue on October 11, 2017 at 1:43 am

    HI, Shoba,
    Thanks for sharing. I am pretty headache about a false positive PCR amplification my technician is experiencing. I suspect the contamination of my thermocycler, and want to use 10-20 fold diluted household bleach to decontaminate the hole of the PCR tube holder of the equipment. Can you share how you clean the tube holder of the thermocycler?

    I plan to wipe the hole with a Q-tip wet with bleach, and then wipe it with Q-tip web with water. Any comments?

    Thanks a lot.

  6. Suzanne on April 30, 2010 at 2:27 pm

    Thanks Matt. I just fixed it.
    Thanks for reading!!
    Suzanne

  7. matt on April 30, 2010 at 1:11 pm

    the link to the Prince et al Article is wrong. Should be:
    http://www.ncbi.nlm.nih.gov/pubmed/1571142

  8. John Mackay on April 11, 2010 at 9:02 pm

    Good stuff Shoba. I wouldn’t let chlorine sit on workstations’ stainless steel though. . we spray, wipe and then follow with 70% ethanol to avoid any chlorine pitting issues. But yes, we wipe it on benches and leave it before the 70% wipe.
    One of the issues for us is the actual DNAcidal (?) stability – as you say, its not that stable and I know others use 3% H2O2 which *is* more stable. Would be interesting to know how long it *does* nuke the DNA.

    Best example of the joys of contamination is the intro to the often-cited Kowk and Higuchi paper. Dropping your 100ul reaction (well this was about 1990!) into an Olympic size swimming pool (I add – maybe swimming around a bit to mix it) and then taking out your 100ul again. . .you’d have about 400 templates for contamination (memory disclaimer applies!)

  9. MAD scientist on April 9, 2010 at 8:48 am

    Also, barrier tips help prevent cross contamination. A bit more money but worth it.

  10. Peter on April 9, 2010 at 7:43 am

    Shoba,

    You wanted to type “false POSITIVES” instead of “false negatives”, didn’t you?

    http://en.wiktionary.org/wiki/false_negative

    Peter

    • Shoba on April 9, 2010 at 5:23 pm

      Peter, good catch! Thanks for pointing that. I have fixed it now 🙂

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