General Laboratory Considerations

Every precaution should be taken to avoid contamination of reagents with unknown/unwanted DNA.
This includes the following:

  • Use a “clean area” for the setup of PCR reactions.2 A clean area is a separate lab space (such as a hood) preferably in a separate lab, both of which are free from PCR products. The clean area should contain dedicated lab coats and pipettes (see below) and should be cleansed (either with 10% bleach or UV lamp) after each use. Amplified PCR products should never enter a clean area.
  • Transfer amplified PCR reactions to another area for analysis.
  • Use sterile, aerosol barrier pipette tips to minimize the risk of aerosol contamination. Change tips after each single use.
  • Change gloves frequently, especially after handling DNA.

Procedure

Amplification Procedure
The optimal conditions for the concentration of MTP Taq DNA Polymerase, template DNA, primers, and MgCl2 will depend on the system being utilized. It may be necessary to determine the optimal conditions for each individual component. It is recommended that the enzyme and the MgCl2 be titrated to determine the optimal efficiency if the below protocol is shown to be less than satisfactory. Sigma offers a separate PCR Optimization Kit, Catalog Number OPT2, that contains a variety of buffers and adjuncts for optimizing the specificity, fidelity, and yield of a PCR product.


1. Add the following reagents to a suitable PCR tube/plate in the following order:

*Maintain final concentrations when scaling reaction volumes. Preparing enzyme / nucleotide / buffer master mixes either with or without primers or template facilitates the setup of multiple reactions.

2. Vortex gently to mix. Briefly centrifuge to collect reaction on the bottom of the tube.

3. If using a thermal cycler without a heated lid, add 50 µL of mineral oil to the top of each tube to prevent evaporation.

4. Cycle using thermocycler of choice. The amplification parameters may require optimization for individual primers, templates, and thermal cyclers.

Common cycling parameters are:

5. Amplified DNA can be evaluated by standard methods (e.g. agarose gel electrophoresis).3 If used, mineral oil may be removed by a single 50 µL chloroform extraction.

Materials
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References

1.
Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski EB, Fox AJ. 2000. Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR. 38(5):1747-1752. http://dx.doi.org/10.1128/jcm.38.5.1747-1752.2000
2.
Millar BC, Xu J, Moore JE. 2002. Risk Assessment Models and Contamination Management: Implications for Broad-Range Ribosomal DNA PCR as a Diagnostic Tool in Medical Bacteriology. Journal of Clinical Microbiology. 40(5):1575-1580. http://dx.doi.org/10.1128/jcm.40.5.1575-1580.2002
3.
Sambrook J, Russel D. 2001. Molecular Cloning: A Laboratory Manual. Third . New York: Cold Spring Harbor Laboratory Press.
4.
Rychlik W, Rhoads RE. 1989. A computer program for choosing optimal oligonudeotides for filter hybridization, sequencing andin vitroamplification of DNA. Nucl Acids Res. 17(21):8543-8551. http://dx.doi.org/10.1093/nar/17.21.8543
5.
Newton C. 1995. PCR: Essential Data. The University of Michigan, New York: John Wiley & Sons p-216.
6.
Choi J, Kim J, Joe C, Kim S, Ha K, Park Y. 1999. Improved cycle sequencing of GC-rich DNA template. Exp Mol Med. 31(1):20-24. http://dx.doi.org/10.1038/emm.1999.3
7.
Rees WA, Yager TD, Korte J, Von Hippel PH. 1993. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry. 32(1):137-144. http://dx.doi.org/10.1021/bi00052a019
8.
Don R, Cox PT, Wainwright B, Baker K, Mattick JS. 1991. ?Touchdown? PCR to circumvent spurious priming during gene amplification. Nucl Acids Res. 19(14):4008-4008. http://dx.doi.org/10.1093/nar/19.14.4008

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No license is conveyed with the purchase of this product under any of US Patents Nos. 5,804,375, 5,994,056, 6,171,785, 6,214,979, 5,538,848, 5,723,591, 5,876,930, 6,030,787, and 6,258,569, and corresponding patents outside the United States, or any other patents or patent applications, relating to the 5’ Nuclease and dsDNA-Binding Dye Processes. For further information contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.