PCR Contamination issues - PCR false positives (Oct/01/2004 )
I am stuck with my PCR work for nearly 1 month due to contamination (false positives even when I set my reactions only with water). I already changed all stocks including primers, washed pipettes with bleach, I use filter-tips and wear gloves, perform pre and post-PCR operations away from where PCR is carried out. I even UV-irradiated the thermocycler for more than 3 hours. Can anyone help me?
Thank you in advance for the slightest hint.
Don't forget the water! Are you using disposable PCR tubes?
SVTX
while making mastermix for pcr try pipetting in all reagents along sides of pcr tubes rather than going down to the end of tube. later spin down all stuff on benchtop spin
also pipette slowly rather than just aspirating all reagents quickly
i have had worse situation than u
PCR contaminations are like ghosts... you know they are there but you dont know WHERE??
I have followed the following with good results...
1. 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.
2. Use a separate aliquot of DEPC water stock for each round of PCR
3. 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.
4. Use a different pipette tip when pipetting all your reagents, even the same master mix to each tube
5. 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.
6. More important... schedule your PCR when not handling plasmids!!!
Hope this help...
Good luck
it could be your gloves...an honors student in a lab near us had the same problem where she changed all her reagents and even used our buffers and dNTP's etc . there was no contaminatin in our buffers or primers etc..eventually her supervisor found out it was her gloves..
if you make up your master mix first, change your gloves after you load your samples. if you load your samples before you master mix then change your gloves just before you make up your master mix.
hope it heaps
is the band in the false positive very faint??? or is it similar in intensity
when compared with ur positive control?? If it is faint in case of false positive and very bright in case of positive control then you can also try reducing the number of cycles.
good luck!!!
Using fewer cycles won't get rid of contamination although it gives a false impression that there is no contamination.
SVTX
thanks for your work Thank you all, apparently contamination disappeared as it came. I only wish it never ever happens again. I keep UV-radiating every stock except primers and taq and follow each of your tips. Thank you again for support...