Standard PCR protocol

Introduction

Taq DNA Polymerase is a thermostable enzyme derived from the thermophilic bacterium Thermus aquaticus. The enzyme is in a recombinant form, expressed in E. coli. It is able to withstand repeated heating to 95 °C without significant loss of activity. The enzyme is approximately 94 kDa by SDS-PAGE with no detectable endonuclease or exonuclease activity. It has 5'→3' DNA polymerase activity and 5'→3' exonuclease activity. Each lot of Taq DNA Polymerase is tested for PCR amplification and double-stranded sequencing. The enzyme is supplied at 5 units/µL and comes with an optimized 10x reaction buffer.

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

Reagents

Select Taq DNA polymerase based upon user’s preference:


Standard Taq DNA Polymerase
Containing MgCl2 Separate MgCl2 Readymix- premixed PCR mastermix with buffer and dNTPs
Clear formulation without dye With red dye for direct load on gels Clear formulation without dye With red dye for direct load on gels Clear formulation without dye
Taq DNA Polymerase from Thermus aquaticus (D1806) REDTaq® DNA Polymerase
(D4309)
Taq DNA Polymerase from Thermus aquaticus, without MgCl2 (D4545) REDTaq® ReadyMix™ PCR Reaction Mix
(R2523)
ReadyMix™ Taq PCR Reaction Mix
(P4600)


·         PCR grade water (W1754)
·         Primers diluted to working concentration (10µM working stocks are sufficient for most assays)
·         Oligos → Order Custom Oligos here
·         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:
    ·         Individual thin-walled 200 µL PCR tubes (Z374873 or P3114)
    ·         Strip tubes, 200uL (Z374962)
    ·         Plates
        ·         96 well plates (Z374903)
        ·         384 well plates (Z374911)
    ·         Plate seals
        ·         AlumaSeal® 96 film (Z721549)
·         dNTP mix, 10 mM each of dATP, dCTP, dGTP, and dTTP (D7295, not needed for readymixes)

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 Taq DNA polymerase, cycling parameters, and the MgCl2 concentration. It is recommended the enzyme and the MgCl2 be titrated to determine the optimal efficiency.


1. Select appropriate table for reaction setup: standard or readymix reagent. Add the reagents to a appropriate sized tube in the order provided in the table. For large number of reactions, a mastermix without the template should be setup and aliquoted into reaction tubes. At the end, template should be added to appropriate tubes.

Standard PCR reaction

Amount Component Final Concentration
w µL Water  
5 µL 10x PCR Buffer (P2192, B5925 or P2317)* 1x
1 µL* Deoxynucleotide Mix 200 µM
w µL Forward primer
(typically 15-30 bases in length)
0.1-0.5 µM
x µL
Reverse primer
(typically 15-30 bases in length)
0.1-0.5 µM
0.5 µL
Taq DNA Polymerase (D6677 or D5684)*
0.05 units/µL
y µL
Template DNA (typically 10 ng)
200 pg/µL
z µL
25 mM MgCl2 (use only with buffer P2317)
0.1-0.5 mM
50 µL
Total reaction volume

*Note: Components listed are reagents sold as D1806, D4309 or D4545

Readymix PCR reaction

Amount Component
Final Concentration
25 µL
Readymix (R2523 or P4600)
 
w µL
Forward primer
(typically 15-30 bases in length)
0.1-0.5 µM
x µL
Reverse primer
(typically 15-30 bases in length)
0.1-0.5 µM
y µL
Template DNA (typically 10 ng)
200 pg/µL
z µL
Water
 
50 µL
Total reaction volume


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.

Typical cycling parameters:

25-30 cycles of amplification are recommended
Denature template
94 °C
1min
Anneal primers
55 °C
2 min
Extension
72 °C
3 min


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.

References

  1. Cheng, S., et al.,  Proc. Natl. Acad. Sci. USA, 91, 5695-5699 (1994).
  2. Chou, Q., Nucleic Acids Res. 20, 4371 (1992).
  3. Innis, M.A., et al. (Eds.)  PCR Strategies, Academic Press, New York (1995).
  4. Innis, M., et al. (Eds.)  PCR Protocols:  A Guide to Methods and Applications, Academic Press, San Diego, California (1990).
  5. Innis, M., et al., Proc. Natl. Acad. Sci. USA 85, 9436-9440 (1988).
  6. Newton, C.R.  (Ed.)  PCR: Essential Data, John Wiley & Sons, New York (1995).
  7. Olive, D., et al., J. Clin. Microbiol. USA 27, 1238 (1989).
  8. Paabo, S., et al., Nucleic Acids Res. 16, 9775-9787 (1988).
  9. Saiki, R., PCR Technology: Principles and Applications for DNA Amplification, Stockton, New York (1989).
  10. Sambrook, J, et al.  Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, New York (2000) . Catalog No. M8265.
  11. Sarkar, G., et al., Nucleic Acids Res. 18, 7465 (1990).
  12. Winship, P.R., et al., Nucleic Acids Res. 17, 1266 (1989).

 

Label License Statement

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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