Biology Reference
In-Depth Information
Autoclaving may not eliminate DNA contamination (
Dwyer and Saksena
1992
). In fact, PCR protocols published by the Cold Spring Harbor Laboratory
recommend using microfuge tubes and tips without autoclaving them first to
reduce the likelihood that undegraded DNA left over from previous autoclave
cycles will contaminate them (
Sambrook and Russell 2001
).
It is
crucial
to separate physically the PCR amplification site from the location
where the PCR products are evaluated by electrophoresis. Ideally, three separate
sites, or rooms, will be available: one for DNA extraction, one for PCR amplifica-
tion, and one for analysis of PCR products. Each separate room or containment
unit should have a
separate
set of supplies and pipettors. Amplified DNA should
never be brought into the area where DNA is being prepared for amplification
or where it is being extracted. Reagents and supplies should never be taken
from an area where PCR analyses are performed.
PCR reagents should be aliquotted to minimize the possibility of contamina-
tion. All reagents should be prepared, aliquotted, and stored in an area free of
PCR products. Similarly, primers should be synthesized and purified in an envi-
ronment free of PCR products.
To reduce contamination from barrels of pipettors, use positive displacement
pipettors with disposable tips and plungers that are completely self contained.
Don't “shoot” the tips off after use; that helps to make an aerosol of the DNA.
Gently pull tips off the pipettor, especially after handling PCR products. Tips that
are plugged with a filter should reduce contamination from DNA aerosolization.
Contamination also can come from electrophoresis equipment, dot-blot appara-
tus, razor blades, microcentrifuges, water baths, and other equipment.
Contamination risks can be reduced by changing gloves frequently (especially
between DNA extraction, PCR amplification, and analysis), wearing different
laboratory coats for DNA preparation, PCR amplification and analysis, uncap-
ping tubes carefully to reduce aerosol formation, and minimizing handling of
DNA samples (
Kitchin et al. 1990
). Components of the PCR (mineral oil, dNTPs,
primers, buffer, and enzyme) can be added to the tubes before adding the tem-
plate DNA. Contamination will be reduced if each tube is capped before adding
DNA to the next.
The use of positive controls can create a contamination problem. Ideally, if a
positive control
is necessary to demonstrate that your PCR is working appropri-
ately, it should consist of template DNA that amplifies
weakly
, but consistently.
Using DNA that produces strong positive responses will generate large amounts
of amplified DNA, which is likely to cause contamination problems. It may be
