biotechrabbit™ YourTaq Hot Start DNA Polymerase is optimized for high yield of amplification of 0.1–3 kb DNA targets, even from low copy number. The enzyme shows excellent PCR specificity and sensitivity for a broad range of amplicons. YourTaq Hot Start DNA Polymerase is resistant to PCR inhibitors, such as blood (up to 20%), Ethanol or humic acid enabling PCR amplification from DNA templates with carry-over of PCR-inhibitors.

YourTaq Hot Start DNA Polymerase is a highly pure enzyme. Together with the optimized buffer and high-quality dNTPs (BR0600202) a mix is achieved for the most demanding PCR applications. In addition, the enzyme is suitable for amplification of GC-rich templates (up to 70%) pairing with 5X PCR Enhancer (BR1900201).

Info: Recommended annealing temperature is 2°C above primer Tm (use gradient PCR to optimize the annealing temperature).

Unit Definition

One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmol of dNTP into acid-insoluble form in 30 minutes at 72°C in the presence of the reaction buffer.

Quality Control

Functional assay

Human genomic DNA was amplified using the DNA Polymerase and specific primers to produce a distinct band of 750 bp.

Self-priming activity

Standard PCR is carried out without primers, using the DNA Polymerase and human genomic DNA. No products were amplified.

Exonuclease assay

Linearized lambda/HindII fragments are incubated with the DNA Polymerase in a 50 µl reaction mixture for 4 h at 37°C. No degradation of DNA was observed.

Endonuclease assay

lambda DNA is incubated with the DNA Polymerase in a 50 µl reaction mixture for 4 h at 37°C. No degradation of DNA was observed.

Nick Activity

Supercoiled plasmid DNA is incubated with the DNA Polymerase in a 50 µl reaction mixture for 4 h at 37°C. No conversion of covalently closed circular DNA to nicked DNA was detected.

E. coli DNA contamination assay

A sample of the denatured DNA Polymerase is analyzed with specific primers targeting the 16S rRNA gene in qPCR for the presence of contaminating E. coli DNA. No E. coli DNA was detectable.

Prevention of PCR contamination

When assembling the amplification reactions, care should be taken to eliminate the possibility of contamination with undesired DNA.

• Use separate clean areas for preparation of samples and reaction mixtures and for cycling.
• Wear fresh gloves. Use sterile tubes and pipette tips with aerosol filters for PCR setup.
• Use only water and reagents that are free of DNA and nucleases.
• With every PCR setup, perform a contamination control reaction that does not include template DNA.

Standard PCR setup

The standard PCR protocol using biotechrabbit reaction buffer provides excellent results for most applications. Optimization might be necessary for certain conditions, such as high GC or AT content, strong template secondary structures or insufficient template purity. In such cases, optimization of template purification (see biotechrabbit nucleic acid purification kits), primer design and annealing temperature is recommended.

The best conditions for each primer-template can be optimized with the following:

• Choosing the optimal quantities of template and primers
• Determining optimal concentrations of the enzyme and magnesium ions
• Optimizing cycling conditions

If unspecific amplification occurs, the amount of DNA Polymerase and the primer concentration can be reduced. Correspondingly, these can be increased when yield is low.

Optimizing magnesium concentration

Many applications use the standard concentration of 1.5–2 mM MgCl₂. However, reactions with increased amounts of template (e.g., genomic DNA), primer and nucleotides might require higher MgCl₂ concentrations (2–3 mM). A separate 50 mM MgCl₂ solution is supplied with the enzyme and can be used to adjust the MgCl₂ concentration according to the table below:

Basic Protocol

• Thaw on ice and mix all reagents well, especially the MgCl₂ solution and dNTPs.
• Keep all reagents and reactions on ice.
• When setting up multiple reactions, prepare a master mix of water, buffer, dNTPs and polymerase. Prepare enough master mix for one more than the actual number reactions. Alternatively, use biotechrabbit 2X YourTaq Hot Start PCR Master Mix (cat. no. BR0202201)
• Pipet the master mix into thin-walled 0.2 ml PCR tubes.
• Add template and primers separately if they are not used in all reactions.

• For total reaction volumes other than 50 µl, scale reagents proportionally.
• Mix and centrifuge briefly to collect the liquid in the bottom of the tube.
• Place in the PCR cycler.

Cycling Program

• Add loading dye solution (see 6X DNA Loading Dye, BR0800301) to the reactions to analyze PCR products on a gel or store them at −20°C.

Product manual

Other documentation