John M. S. Bartlett.pdf - Bio-Nica.info

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stored. This means that that they can be identified and discarded if contamination
does arise (9). Master mixes are made up here and added to reaction vessels before
continuing onto the next stage of the process (see Note 5). If necessary, an oil overlay
can also be added at this stage.
3.2. Sample Preparation Laboratory
Laminar flow cabinets are necessary for dealing with samples until they are
inactivated and extracted, and these and other equipment should be decontaminated
before and after each procedure (see Note 3). The equipment necessary will depend on
the extraction methods used, but a microfuge, heating block, and vortex are minimal
requirements.
3.3. PCR Laboratory
Primary and secondary PCR steps should be separated, preferably in different rooms,
and certainly with separate thermocyclers; however, the layout of this area will depend
on space and equipment available. Primary reactions containing master mix and nucleic
acid should be assembled and placed on the appropriate thermocycler. After cycling,
these are removed to the secondary PCR area, where reactions are assembled and
placed on cyclers dedicated for this process. Other automated/integrated/single-round
equipment should be positioned with secondary thermocyclers to reduce the risk of
contamination (see Note 6).
3.4. Post-PCR Processing
All final amplified products should be dealt with in this area, which can be used
for techniques, including electrophoresis, restriction fragment length polymorphism
(RFLP), hybridization work, cloning, and sequencing. It is important that nothing from
this area should go back through other areas involving preliminary steps but should be
processed through a waste management or discard area.
4. Notes
1. For example, hot blocks are easier to decontaminate on a regular basis and are therefore
a better option than water baths.
2. Normal tips can be used for post-PCR steps.
3. An ultraviolet irradiation source is valuable in reducing contamination; however, Cimino
et al. (10) recommend caution when using this method alone. Otherwise, wash down all
nonmetal surfaces with 0.1 N HCl, or 10% bleach, followed by water.
4. Nitrile gloves should be used for safety when handling ethidium bromide if used in gel
electrophoresis.
5. As kit-based formats become available, reagent and master mix will be supplied, completely
reducing the need for this area.
6. This setup will become more difficult as combined extraction/amplification and detection
equipment become more available.
References
1. Wolcott, M. J. (1992) Nucleic acid-based detection methods. Clin. Microbiol. Rev. 5,
370–386.
2. Wilson, I. G. (1997) Inhibition and facilitation of nucleic acid amplification. Appl. Environ.
Microbiol. 63, 3741–3751.