Procedure

Note: The use of DMSO or formamide with JumpStart REDTaq DNA Polymerase is not recommended due to interference with the enzyme-antibody complex. Other co-solvents, solutes (e.g., salts) and extremes in pH or other reaction conditions may reduce the affinity of the JumpStart Taq antibody for the Taq DNA polymerase and thereby compromise its effectiveness.

Preparation of PCR Master Mix and Thermal Cycling Parameters

Because Taq DNA polymerase is a magnesium ion-dependent enzyme, the optimal conditions for the concentration of Taq, 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. This is especially true for JumpStart REDTaq, cycling parameters, and the MgCl2 concentration. It is recommended the enzyme and the MgCl2 be titrated to determine the optimal efficiency.

To minimize tube-to-tube variation, preparation of a PCR master mix with JumpStart REDTaq DNA Polymerase is recommended. The amount prepared should be based on the number of PCR reactions to be performed.

1. Add the following reagents to a 0.2 mL or 0.5 mL PCR tube.

*The individual nucleotides (1 µL of each 10 mM solution, 4 µL total) may be replaced by 1 µL of Deoxynucleotide Mix, Catalog Number D7295.

2. Mix gently and briefly centrifuge to collect all solution at the bottom of the tube.

3. Add 50 µL of mineral oil to the top of each tube to prevent evaporation (optional, depending on model of thermal cycler).

4. Amplification parameters will vary depending on the primers and the thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler.

Typical cycling parameters:

* 1 minute minimum or 1 minute per kb expected amplicon.

5. The amplified DNA can be evaluated by loading 5-10 µL of the PCR reaction directly onto agarose gel. It is not necessary to add a separate loading buffer/tracking dye.

Note: A minimum of 1.5 units of JumpStart REDTaq DNA polymerase must be added per 50 µ reaction to ensure enough glycerol is present for direct gel loading. The red tracer comigrates with 125 bp fragment in a 1% agarose gel.

Troubleshooting Guide

Materials
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References

1.
Griffin H, Griffin A. 1994. PCR Technology: Current Innovations. CRC Press.
2.
Kwok S, Higuchi R. 1989. Avoiding false positives with PCR. Nature. 339(6221):237-238. http://dx.doi.org/10.1038/339237a0
3.
Innis M. 1995. PCR Strategies. New York: Academic Press.
4.
Innis M. 1990. PCR Protocols: A Guide to Methods and Applications. San Diego, California: Academic Press.
5.
Newton C. 1995. PCR: Essential Data. New York: John Wiley & Sons.
6.
Sambrook J. 2000. Molecular Cloning: A Laboratory Manual. Third Edition. New York: Cold Spring Harbor Laboratory Press.
7.
Huang L, Jeang K. 1994. BioTechniques. 16242-246.

NOTICE TO PURCHASER: DISCLAIMER OF LICENSE

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 5â 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.

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