Every precaution should be taken to avoid contamination of reagents with unknown/unwanted DNA.
This includes the following:
The optimal conditions for the concentration of MTP 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. It is recommended that the enzyme and the MgCl2 be titrated to determine the optimal efficiency if the below protocol is shown to be less than satisfactory. Sigma offers a separate PCR Optimization Kit, Catalog Number OPT2, that contains a variety of buffers and adjuncts for optimizing the specificity, fidelity, and yield of a PCR product.
1. Add the following reagents to a suitable PCR tube/plate in the following order:
*Maintain final concentrations when scaling reaction volumes. Preparing enzyme / nucleotide / buffer master mixes either with or without primers or template facilitates the setup of multiple reactions.
2. Vortex gently to mix. Briefly centrifuge to collect reaction on the bottom of the tube.
3. If using a thermal cycler without a heated lid, add 50 µL of mineral oil to the top of each tube to prevent evaporation.
4. Cycle using thermocycler of choice. The amplification parameters may require optimization for individual primers, templates, and thermal cyclers.
Common cycling parameters are:
5. Amplified DNA can be evaluated by standard methods (e.g. agarose gel electrophoresis).3 If used, mineral oil may be removed by a single 50 µL chloroform extraction.
JumpStart is a trademark of Sigma-Aldrich Co. LLC
MTP is a trademark of Sigma-Aldrich Co. LLC
Restorase is a registered trademark of Sigma-Aldrich Co. LLC
TWEEN is a registered trademark of Croda International PLC
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.