Procedure

Note: JumpStart Taq DNA polymerase has been shown to work effectively with up to 5% v/v DMSO. 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 the 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 the JumpStart Taq, 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 Taq is recommended. The amount prepared should be based on the number of PCR reactions to be performed.

1. For a single reaction, add the following reagents to a 0.2 or 0.5 mL microtubes in the following order:

*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 the 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 agarose gel electrophoresis and subsequent ethidium bromide staining. Mineral oil overlay may be removed by a single chloroform extraction (1:1), recovering the aqueous phase.

Troubleshooting Guide

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References

1.
HUANG L, JEANG K. 1994. Long-range jumping of incompletely extended polymerase chain fragments generates unexpected products. Biotechniques.. 16(2):242-6.
2.
Kwok S, Higuchi R. 1989. Avoiding false positives with PCR. Nature. 339(6221):237-238. http://dx.doi.org/10.1038/339237a0
3.
Griffin H, Griffin A. 1994. PCR Technology: Current Innovations. CRC Press.
4.
Innis M. 1995. PCR Strategies. New York: Academic Press.
5.
Innis M. 1990. PCR Protocols: A Guide to Methods and Applications. San Diego, California. Academic Press.
6.
Newton C. 1995. PCR: Essential Data. New York: John Wiley & Sons.
7.
Sambrook J. 2000. Molecular Cloning: A Laboratory Manual. Third Edition. New York: Cold Spring Harbor Laboratory Press.

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