7/30/2019 Guidelines Preventing Contamination Pcr

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Protocol

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Guidelines for Preventing

Contamination of PCR

During PCR more than 10 million copies o a template

DNA are generated. Thereore, care must be taken to

avoid contamination with other templates and amplicons

that may be present in the laboratory environment.General recommendations to lower the risk o

contamination are the ollowing:

Prepare your DNA sample, set up the PCR mixture,

perorm thermal cycling and analyze PCR products in

separate areas.

Set up mixtures or PCR in a laminar ow cabinet

equipped with an UV lamp.

Wear resh gloves or DNA purifcation and reaction

set up.

Use containers dedicated or PCR. Use positive

displacement pipettes, or use pipette tips with aerosol

flters to prepare DNA samples and set up PCR.

Use certifed reagents, including high quality water

(e.g., Water, nuclease-ree).

Always perorm No-Template-Control (NTC) reactions

to check or the absence o contamination.

For detailed instructions or the set-up o a PCR

laboratory and its maintenance, reer to PCR Methods

and Applications, 3, 2, S1-S14, 1993.

PCR requently is contaminated by amplicons rom

previous PCR held in the same room. One o the most

popular and efcient methods or prevention o carryover

contamination is a use o uracil DNA glycosylase* (UDG)(1). A part or all o the dTTP in the PCR reaction is

substituted by dUTP and thereore all PCR products

generated in your working environment contain dUTP.

Prior to each PCR, short incubation with UDG eliminates

such contaminating amplicons carried over rom the

previous PCR. Incorporation o dUTP does not aect the

intensity o ethidium bromide staining or the electropho-

retic mobility o the PCR product, thereore the reactions

can be analyzed by standard agarose gel electrophoresis.

Taq DNA polymerase and all other non-prooreading

polymerases will incorporate dUTP into a PCR product,

but prooreading polymerases or enzyme mixes containingsuch prooreading polymerases (e.g, DreamTaq DNA

Polymerase, High Fidelity Enzyme Mix or the Long

PCR Enzyme Mix), do not incorporate dUTP or may

incorporate with much less efciency.

* Use o such enzyme in certain territories may be covered

by patents and may require a license.

Reference

1. Longo, M.C., et al., Use o uracil DNA glycosylase

to control carry-over contamination in polymerase

chain reactions, Gene 93, 125-8, 1990.

This protocol is for the Guidelines for Preventing Contamination of PCR