HotStarTaq DNA Polymerase

Touchdown PCR uses a cycling program with varying annealing temperatures. It is a useful method to increase the specificity of PCR. The annealing temperature in the initial cycle should be 5–10°C above the Tm of the primers. In subsequent cycles, the annealing temperature is decreased in steps of 1–2°C/cycle until a temperature is reached that is equal to, or 2–5°C below, the Tm of the primers. Touchdown PCR enhances the specificity of the initial primer–template duplex formation and hence the specificity of the final PCR product.

To program your thermal cycler for touchdown PCR, you should refer to the manufacturer’s instructions. For additional hints and tips for successful PCR, review the Appendix Sections in our PCR Kit handbooks, and our Brochures and Application Guides for PCR and RT-PCR.

Both the quality and quantity of nucleic acid starting template affect PCR, in particular the sensitivity and efficiency of amplification. PCR sensitivity and efficiency can be reduced by the presence of impurities in nucleic acid preparations or in biological samples. These PCR inhibitors are completely removed when template is prepared using QIAGEN Kits for nucleic acid purification. Please refer to the Brochure "Maximizing PCR and RT-PCR success" for additional information.

The optimal primer–template ratio has to be determined empirically. If too little template is used, primers may not be able to find their complementary sequences. Too much template may lead to an increase in mispriming events. Generally, no more than 1 ug of template DNA should be used per PCR reaction. As an initial guide, spectrophotometric and molar conversion values for different nucleic acid templates are listed below.

Spectrophotometric conversions for nucleic acid templates

*Absorbance at 260 nm = 1

Molar conversions for nucleic acid templates

* Base pairs per haploid genome

** For single-copy genes

PCR products that will be cloned using the QIAGEN PCR Cloning Kit should be generated using a thermostable DNA Polymerase without proofreading activity, such as Taq DNA Polymerase. Such polymerases attach a single A overhang to their reaction products, which can hybridize to the U overhang of the pDrive Cloning Vector. For efficient addition of an A overhang during the PCR procedure, we recommend a final extension step for 10 min at 72°C as described in the standard protocols of the Taq PCR- and HotStarTaq PCR handbook.

Please see the following factors that can contribute to unspecific, smeared PCR products, and suggestions how to avoid it:

• too much starting template

  Check the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions.

• carry-over contamination

  If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable pipet tips containing hydrophobic filters to minimize cross-contamination. Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.

• enzyme concentration too high

  When using HotStarTaq or Taq DNA Polymerase, use 2.5 units per 100 µl reaction.

• too many PCR cycles

  Reduce the number of cycles in steps of 3 cycles.

• Mg2+ concentration not optimal

  Perform PCR with different final concentrations of Mg2+ from 1.5–5.0 mM (in 0.5 mM steps) using the 25 mM MgCl2 solution provided (see table below):

• Primer concentration not optimal or primers degraded

  Repeat the PCR with different primer concentrations from 0.1–0.5 µM of each primer (in 0.1 µM steps). In particular, when performing highly sensitive PCR, check for possible degradation of the primers on a denaturing polyacrylamide gel.

• Primer design not optimal

  Review your primer design, and design new primers

For additional information on optimization of PCR results, please refer to the Appendix sections of the Taq PCR and HotStarTaq DNA Polymerase Handbook, and our comprehensive Brochure Critical Factors for Successful PCR.