HotStarTaq DNA Polymerase (2024)

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, andour Brochures and Application Guidesfor 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 ratiohas tobedetermined 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 Polymerasewithout proofreading activity, such as Taq DNA Polymerase. Such polymerases attach a single A overhang to theirreaction 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 protocolsof theTaqPCR- and HotStarTaq PCR handbook.

Please see the followingfactors thatcan 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 HotStarTaqor 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.

No, we do not sell Q-Solution separately. It is available only as a component of the Taq DNA Polymerase, Taq PCR Core, HotStarTaq DNA Polymerase, QIAGEN Multiplex PCR-, and the QIAGEN OneStep RT-PCR Kits.

CoralLoad dyes supplied in PCR Kits such as, e.g.,Taq, HotStarTaq, and TopTaq DNA Polymerase and TopTaq Master Mix do not interfere with most downstream enzymatic applications.

However, for reproducible results, purification of PCR products using the QIAquick or MinElute PCR Purification Kits prior to enzymatic manipulation is recommended.

Yes, please follow the Supplementary Protocol 'Polyacrylamide_gel_analysis_of_oligonucleotides'(PCR03).

Not necessarily. In a lot of cases, the uniquely formulated PCR Buffer providedin the HotStarTag PlusDNA Polymerase, HotStarHiFidelity Polymerase,Taq DNA Polymerase, HotStarTaq DNA Polymerase,and QIAGEN Multiplex PCR Kitsprovides optimal amplification of specific PCR products. The usefulnessof Q-Solution needs to be determined empirically for each primer/template setup, by running parallel PCR reactions with and without Q-Solution under the same cycling conditions.

Q-Solution changes the melting behavior of DNA andwill oftenimprove a suboptimal PCR caused by templates that have ahigh degree of secondary structure or high GC-contents. For more details on the effects of Q-Solution on PCR amplification, please see the Q-Solution sectionsof the HotStarTaq Plus DNA Polymerase, HotStar HiFidelity Polymerase, Taq DNA Polymerase, HotStarTaq DNA Polymerase, and the QIAGEN Multiplex PCR Handbooks.

Yes. Please seeTable 3 in our brochure Maximizing PCR and RT-PCR success. We tested the effects of different inhibitory substances in a number of PCR systems. We also analyzed the effect of including different volumes of reverse transcription (RT) reaction mixtures in PCR. Please see the table below for alist of commonly encountered templateimpurities and theirinhibitory effects on PCR.

Impurities showing inhibitory effects on PCR

QIAGEN's 10x PCR Buffer providedin the Taq DNA Polymerase, Taq PCR Core, and HotStarTaq DNA Polymerase Kits contains:

• Tris-Cl
• KCl
• (NH₄)₂SO₄
• 15 mM MgCl₂ ; at pH 8.7 (20°C).

Note that further details on the composition of the 10x PCR Buffer are proprietary.

Todetermine the optimal annealing temperature for a PCR assay, a Temperature Gradientexperiment should be performed. To do this, you will set up several PCR reactions in duplicate for the same primer/template combination, using the same PCR chemistry, and subject each of the reactions to a slightly different annealing temperature within a specified range. If a thermal cycler with a temperature gradient function can be used, you can simply program a temperature rangefor adjacent wells in the cycling block. If no cycler with a gradient function exists in your lab, you will either have to perform duplicate reactions at different temperatures indifferent machines (if available), or back to back in the same machine.