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Amplification of DNA Using Jumpstart™ REDTaq® DNA Polymerase (D8187)

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.

Volume Reagent Final Concentration
5 µL 10x PCR Buffer 1x
1 µL* 10 mM dATP 200 µM
1 µL* 10 mM dCTP 200 µM
1 µL* 10 mM dGTP 200 µM
1 µL* 10 mM TTP 200 µM
- µL Primers 0.1-0.5 µM
2.5 µL JumpStart REDTaq DNA Polymerase 0.05 units/µL
- µL Template DNA (typically 10 ng) 200 pg/µL

Water -
50 µL Total reaction volume

*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:

Initial denaturation 94 °C for 1 min
25-35 cycles:
Denaturation 94 °C for 30 sec
Annealing 55 °C to 68 °C for 30 sec
Extension 72 °C for 1 min (minimum)*
1 cycle:
Final extension 72 °C for 1 min (minimum)*
Hold 4 °C

* 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.

roubleshooting Guide

Problem Suggestion
No reduction of non-specific products is observed when using JumpStart REDTaq DNA Polymerase. Test the PCR system using a manual hot start method.
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 polymerase and thereby compromise its effectiveness.
Both the JumpStart REDTaq PCR and the manual hot start PCR yield multiple nonspecific products. Raise the annealing temperature in 2-3°C increments. Raising the temperature improves the specificity of binding by the primers; however, it may result in reduced binding and extension of the primers. If raising the annealing temperature causes a reduced yield of the specific product with only a proportional reduction of side reaction products, it may be necessary to redesign the primers.1
Take special precautions to avoid crossover contamination of PCR reactions with both specific and nonspecific PCR products, including primer-dimer artifacts.2
The JumpStart REDTaq PCR yields more non-specific products than conventional hot start PCR. Titration of JumpStart REDTaq may be necessary to achieve the same degree of improvement as with a conventional hot start. This is especially true if the PCR reaction conditions vary from those described in this document. In this case, start with a working solution that has a two- to four-fold higher concentration of JumpStart REDTaq than recommended.
The yield of specific product is low using JumpStart REDTaq. Increase the reaction volume to 150 µL or more.
Increase the number of amplification cycles. If currently using 25-30 cycles, increase the cycle number to 35-40. This should increase yields without significantly increasing side reaction products.
Modify the reaction conditions and/or selection of PCR targets to obtain greater opportunities for PCR priming. For example, increase the denaturation time up to 1-1.5 minutes and/or increase the denaturation temperature to as high as 95° C to overcome denaturation difficulties.
The use of DMSO or formamide with JumpStart REDTaq is not recommended due to interference with the enzyme-antibody complex.

Materials

     

References

  1. Huang, L. M., and Jeang, K.-T., BioTechniques 16, 242-246 (1994).
  2. Kwok, S., and Higuchi, R., Nature 339, 237-238 (1989).

General References

  • Griffin, H. G. and Griffin, A. M. (Eds.) PCR Technology: Current Innovations, (CRC Press, 1994).
  • Innis, M. A., et al. (Eds.) PCR Strategies, (Academic Press, New York, 1995).
  • Innis, M., et al. (Eds.) PCR Protocols: A Guide to Methods and Applications, (Academic Press, San Diego, California, 1990).
  • Newton, C. R. (Ed.) PCR: Essential Data, (John Wiley & Sons, New York, 1995).
  • Sambrook, J. F., et al. Molecular Cloning: A Laboratory Manual, Third Edition, (Cold Spring Harbor Laboratory Press, New York, 2000.

 

JumpStart is a trademark of Sigma-Aldrich Co. LLC
REDTaq is a registered trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.
TWEEN is a registered trademark of Croda International PLC.

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 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.

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