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Equipping, Establishing a PCR Laboratory 15
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Equipping and Establishing a PCR Laboratory
Susan McDonagh
1. Introduction
Polymerase chain reaction (PCR) is a very sensitive method of amplifying specific
nucleic acid, but the system is susceptible to contamination from extraneous or
previously amplified DNA strands (1,2). Many specific copies of DNA are produced
from each round of amplification (3) with a single aerosol containing up to 24,000
copies of amplified material (4). The most important consideration when designing
and equipping a laboratory for PCR is therefore to minimize the risk of contamination
and ensure accurate results (5,6). To do this, it is necessary to physically separate the
different parts of the process and arrange them in a unidirectional workflow (4). This
avoids back flow of traffic and, along with restricted access, will reduce the risk of
contamination and inaccurate results.
The way in which the workflow is arranged will depend on the amount of available
space. If possible, different rooms should be used for reagent preparation, sample
preparation, PCR (some also separate primary and secondary stages), and post-PCR
processing (see Fig. 1). Each of these areas should contain dedicated equipment,
protective clothing, and consumables (1). Disposable gloves should be readily available
for frequent changing to avoid cross contamination, and control material should be
included in every run to monitor any contamination problems (3).
2. Equipment
A list of basic equipment required for a PCR laboratory is given in Table 1.
2.1. Thermocyclers
This is obviously the most important piece of equipment in the laboratory, with
many products available from different manufacturers. Thermocyclers can be supplied
with a variety of reaction vessel formats, including 0.2- and 0.5-mL microtubes; strips
of tubes; microtiter plates containing up to 384 wells; glass slides; and capillaries.
Temperature ramp rates and uniform heat distribution across the block are important
for consistent performance. These options, along with the consideration of laboratory
requirements, are factors when purchasing a machine, and these specifications are obviously
reflected in the cost. For example, if basic PCR is all that is required, equipment
from the lower end of the range might suffice. These machines have programmable
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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