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How to avoid contamination in PCR?

Oct 02 2004 12:24 PM | bioforum in PCR


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

I have followed the following with good results...
  • 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.
  • Use a separate aliquot of DEPC water stock for each round of PCR
  • 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.
  • Use a different pipette tip when pipetting all your reagents, even the same master mix to each tube
  • 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.
  • More important... schedule your PCR when not handling plasmids!!!

Hope this help...
Good luck

10 Comments


perneseblue
Mar 09 2011 09:57 PM
Contamination will occur. It just takes one moment, one droplet.

So design your PCR kit with that in mind.

Aliquot all your PCR reagents. If contamination does occur, you can then discard all your current PCR reagents and take out new uncontaminated reagents.

This is how you should handle PCR contamination.

bananapaper
May 11 2011 06:37 AM
I have problem in PCR contaminations.
I doubt one of my primer that was contaminated
I had check water, pcrbuffer, dntp and Taq.

I don't want to design primer again.

So~can i use UV light to irradiate my reagents(include F and R )???

OR HAVE any other way to remove  

thank you!!

bbbarcelona
Jun 15 2011 01:18 AM
I think you kan irradiate water, magnesium... but not F and R (as mutations interfere PCR!!!)

Importantly, I believe that post-pcr steps should be performed in a different room than pre-pcr steps.

Sherina Kamal
Sep 26 2011 07:17 PM
thanks for the great info.im do new here...never done this work before,,i have a lot of problems handling PCR...at least now i know how to prevent contamination.

piya29
Oct 15 2012 01:36 PM
I did yeast colony PCR but no band at all,does anybody knows is crimson taq useful for yeast colony pcr?

Curtis
Oct 30 2012 12:15 AM
colony pcr is usually very easy. even 25 cycles is enough. we don't boil to break the membrane. just directly add the colony into the pcr tube and run.

BioMiha
Nov 11 2012 06:21 AM
In my opinion the most important things are:
Don't do too many cycles (30 is enough for everything).
Don't open your PCR tubes in the same area as you (or other people for that matter) are doing the PCR preps. The amplicons get aerosolized and then they are everywhere.

Myworkismyplay
Dec 26 2012 09:17 PM
Bananapaper, what is your "proof" of contamination? Non-specific bands, not bands at all, degraded sample?  that will help people give you the best advice.

For non-specific bands, simple things like reducing template and primer concentrations can fix that. Or maybe the Taq has gone bad if you have no bands.

Sometimes if a primer solution/dilution seems to not work, that's the cheapest thing to replace--- $10-20 at most to just get a new tube of it synthesized.

Just curious!

Anoop Bhaskaran
Feb 07 2013 01:44 AM
my template DNA contains phenolic compounds..so am not  getting amplification...What should I do to get amplification?

muhammad ilyas
Apr 26 2013 04:15 AM
what is concentration of taq and MgCl2 will give the best amplification is there any fixed proportion of these two?
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