Saturday, March 28, 2015

PCR Contamination

You carefully set up your PCR, excitedly waited for it to finish, ran your gel and waited for the big reveal. But instead of seeing what you hoped for: A nice clean gel. You see a big fat mess: Extra bands and, most disturbingly, bands in your negative control. So what are you to do? How do you fix this PCR contamination and avoid it in the future?

What is PCR contamination?

The biggest source of PCR contamination is aerosolized PCR products, which are created when you open a tube of or pipette amplified PCR product. Once created, being small, they travel well. And can easily spread all over your bench and equipment, where they can find their way into your next PCR reaction and get amplified. So now that you knowwhat PCR contamination is made of. How do you know if you have it?

Run Negative Controls

We know we should always run negative controls. Yet sometimes we justify not doing it. We come up with reasons like “This experiment always works!”, “I could save on reagents”, “I do not have the time”. But take it from my (bitter) experience: Always run your dang negative control! Without a negative control PCR contamination can lurk undetected for some time, mucking up experiments, wasting your time with troubleshooting, and slowly spreading throughout your lab. So don’t become over confident and don’t justify your laziness, just run your negative controls because THAT is how you will know if you have contamination. A PCR negative control is usually just the normal PCR master mix (polymerase, primers, buffer, nucleotides) but instead of adding template, you add water. This should result in a blank PCR. Therefore if you DO get a band you know you have contamination…somewhere.

Identify Your Contamination Source

So where could this contamination be coming from? Well anywhere really. It could be coming from1) your laboratory environment such as your pipettes, tips, hands, benchtop, centrifuge, etc; or 2) your reagents such as your polymerase, buffer, nucleotides, water, etc.

1) Rule Out Your Laboratory Environment

First thing first, when tracking down PCR contamination, is to remove all possible environmental sources. To do this you should:
Use a 10% bleach solution or DNA-away to wipe down your:
  • Bench top
  • Pipettes
  • Centrifuge
  • Vortexer
  • Racks
  • Thermocyler lid and buttons
Get new:
  • Unopened filter tip boxes
  • Unopened sterile PCR tubes
Correctly assemble your PCR reaction:
  • Wear a dedicated lab coat. You should wear a lab coat dedicated to PCR setup. This should NOT! be the same coat you wear when analyzing your PCR results. This coat should not go anywhere near open tubes of amplified PCR product.
  • Change your gloves frequently. If you leave your PCR setup and dedicated equipment to do anything – get more reagents, answer your phone, use a pen – change your gloves before returning to the setup.
  • Use only dedicated equipment. The pipettes, centrifuge, vortexer you use when setting up a PCR reaction should be dedicated to PCR setup. No amplified PCR products should come anywhere near them.

2) Rule Out Your Reagents

Now that you have minimized the introduction of any new PCR contamination into your PCR assembly, it is time to check your reagents. This means systematically substituted each of your old reagents with a new (previously unopened) reagent and re-running your blank PCR. Whatever substitution(s) removes your contamination bands is the contaminated reagent, which should be discarded.

Avoid Future Contamination

To avoid future contamination, you should do everything above and a few more things:
  • Work in dedicated space. Setup your PCR away from where you analyze PCR results. This is best done in a hood, or at minimum benches away from where you run yours gels.
  • Store PCR reagents and PCR products separately. In the same way that you do not want your PCR analysis to be near where you setup, you should never store PCR product and reagents together. Whenever possible use separate refrigerators.
  • Aliquot. No matter how careful you are, contamination can still happen.  Contamination costs not just time but money, as you must throw out any contaminated reagents. And if that reagent is new that can mean $300 straight into the trashcan! So whenever possible, aliquot your reagents into smaller tubes and only work from one aliquot at a time. Not only will this help you deal with contamination, but it will also prolongs the life of your reagents by reducing their number of freeze/thaw cycle.
  • Store PCR tubes/tips/racks separately. Keep your PCR stuff clean by storing it well away from PCR product. Label all PCR equipment as such. And tell other lab members to respect their designation.
  • Don’t flick your tubes open. Minimize aerosolizing your PCR product by never “flicking” your PCR tubes open. I know it is faster to open your tubes with one hand by flicking the lid open with your thumb, but this is bad form and contributes to your bench’s contamination. Instead take your time and use two hands to carefully open all PCR product tubes.
  • Use a master mixer and add your template last. The less times you handle your template the less opportunity there is for contamination. Therefore you should always set up your PCR reactions using master mixes made with (in order): water, buffer, nucleotides, primers, polymerase, and LAST! template.
  • Train others. The cleanliness in your lab is only as strong as your weakest lab member. Inform others about PCR contamination, and the steps needed to avoid it. Hey, you could even print this article and share it with them.
All of these sentences I copied from :
Aim : notes for my research. I hope by sharing this article it helps other to solve their problem in PCR.

Nurul Ain