Amplification of Genomic DNA using REDTaq DNA Polymerase

Background

REDTaq DNA Polymerase is a convenient package that includes all the necessary components for a PCR reaction except primers, DNA template and water. The formulation has been optimized for amplification of genomic or other complex DNA templates.

REDTaq DNA polymerase is Sigma’s Taq DNA Polymerase mixed with an inert red dye. The dye provides quick recognition of reactions to which enzyme has been added as well as visual confirmation of complete mixing. The enzyme is provided at 1 unit/µL for more accurate volume measurement and less waste. Reactions using REDTaq are formulated as any PCR mixtures. There are no additional reaction preparation steps or protocol changes required.  These formulations allow aliquots (5-10 µL) from the PCR to be directly loaded onto an agarose gel without addition of electrophoresis loading buffers.  The inert dye co‑migrates at the same rate as a 125 bp fragment in a 1% agarose gel. Because a gel loading buffer is not added to the reaction mix, a sample can be reamplified, such as in nested PCR. If necessary, the dye can be removed from the amplicon by routine purification methodologies. The presence of the dye has no effect on automated DNA sequencing, ligase mediated ligations, exonucleolytic PCR product digestion and transformation. Though exceptions may exist, the dye is generally inert in restriction enzyme digestions.

Unit Definition: One unit incorporates 10 nmol of total deoxyribonucleoside triphosphates into acid precipitable DNA in 30 minutes at 74 °C.

Reagents

Select desired PCR reagent:

Genomic DNA Polymerase, with red dye for direct load on gels
Containing MgCl2 Separate MgCl2
With a tube of dNTPs Without a tube of dNTPs Without a tube of dNTPs
REDTaq® SuperPak™ DNA Polymerase (D6063) REDTaq® Genomic DNA Polymerase, with MgCl2 (D8312) REDTaq® Genomic DNA Polymerase, without MgCl2 (D2812)

  • PCR grade water (W1754)
  • Primers diluted to working concentration (10µM working stocks are sufficient for most assays)
  • DNA to be amplified
  • Dedicated pipettes
  • Thermal cycler
  • Sterile filter pipette tips
  • Sterile 1.5 mL screw-top microcentrifuge tubes (such as CLS430909)
  • PCR tubes, select one of the following to match desired format:
  • dNTP mix, 10 mM each of dATP, dCTP, dGTP, and dTTP (D7295, needed for standard format reagents only)

Procedure

The optimal conditions for the concentration of 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. This is especially true for the REDTaq DNA polymerase, cycling parameters, and the MgCl2 concentration.  It is recommended the enzyme and the MgCl2 be titrated to determine the optimal efficiency. 
 
    1.     Add the following reagents to a 0.2 or 0.5 ml PCR tube in the following order:
 

Amount
Component
Final Concentration
q.s.
Water
-
5 µL
10x PCR Buffer
1x
1 µL
10 mM dNTP mix
200 µM of each dNTP
- µL
Forward primer
0.1-0.5 µM
- µL
Reverse primer
0.1-0.5 µM
2.5 µL
REDTaq DNA Polymerase
0.05 units/µL
-  µL
Template DNA (typically 10 ng)
200 pg/µL
50 µL
Total reaction volume
 

           
    Note: when diluted, the PCR buffer provides a final MgCl2 concentration of 1.5mM.  For some application, higher     levels of MgCl2 maybe optimal.  If needed, user can supplement up to 5.5mM additional MgCl2.

    2.     Mix gently by vortex and briefly centrifuge to collect all components to the bottom of the tube.

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

    4.     The 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.
 
        Common cycling parameters:
        a.     Denature the template at 94 °C for 1 minute
        b.    Anneal primers at 55 °C for 2 minutes
        c.     Extension at 72 °C for 3 minutes
 
        25-30 cycles of amplification are recommended.
 
 
    5.     The amplified DNA can be evaluated by agarose gel electrophoresis by loading 5-10 µL of the PCR reaction     onto the gel without the addition of gel loading buffers.
 
    Note: a minimum of 1.5 units of REDTaq DNA polymerase must be added per 50 µL reaction for unencumbered     gel loading. The red tracer co-migrates with 125 bp fragment in a 1% agarose gel. If a more intense tracking dye     is desired, an unused lane can be used to run any common tracking dye.

References

  1. Innis, M. A., et al. (Eds.)  PCR Strategies, Academic Press, New York (1995)
  2. Innis, M., et al. (Eds.)  PCR Protocols:  A Guide to Methods and Applications, Academic Press, San Diego, California (1990)
  3. Innis, M., et al., Proc. Natl. Acad. Sci. USA 85, 9436-9440 (1988).
  4. Mytelka, D. S., and Chamberlin, M. J., Nucleic Acids Res., 24 (14), 2774-2781 (1996).
  5. Newton, C. R.,  (Ed.)  PCR: Essential Data, John Wiley & Sons, New York (1995)
  6. Sambrook, J., et al.  Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, New York (2000)

 

REDTaq is a registered trademark and SuperPak is a trademark of Sigma-Aldrich Biotechnology LP

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.

Materials

     
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