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Quality Control 21
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Quality Control in PCR
David Stirling
1. Introduction
Polymerase chain reaction (PCR), like any laboratory procedure, can be subject
to a range of experimental or procedural error. A clear consideration of where such
potential errors may occur is essential to minimize their impact. Careful quality control
of equipment and reagents is essential.
2. Equipment
The previous chapter dealt with the sort of equipment that is required to perform
PCR. It is commonplace for an individual laboratory to contain many sets of equipment,
each bought from different manufacturers, at different times, and subjected to various
amounts of abuse from students who don’t know any better and laboratory managers
who do! In an ideal world, and any diagnostic or commercial laboratory, each piece of
equipment should be serviced and calibrated on a regular basis, with careful records
being kept of this maintenance. Unfortunately, not every laboratory has funds for fullservice
contracts on all equipment. There are a few fundamental procedures, however,
which will reduce errors from equipment problems.
• Be consistent in the equipment used for any given PCR. If it works on Monday but not
Tuesday, this may simply be to the result of using a different PCR block. Even the most
modern and expensive thermal cyclers deteriorate with age.
• Check pipetting devices on a regular basis (weekly is not excessive) to ensure they pipet
the correct volume. This is easily performed by pipetting and weighing water. Most
manufacturers produce inexpensive service packs for their pipettors.
3. Reagents
As with all laboratory procedures, it generally pays dividends to use high-quality
reagents from reputable suppliers. You may well know someone who brews their
own Taq polymerase in a vat in the garage, but do they control for batch-to-batch
variability?
The design of optimum PCR primers will be discussed later. It is important to
remember, however, that these are single-stranded DNA molecules and are therefore
relatively labile. Repeated freeze/thawing will cause degradation to shorter products,
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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