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Hot Start Taq DNA Polymerase, 5 U/µl

Features

  • High PCR specificity and sensitivity
  • Exceptionally pure Hot Start Taq DNA Polymerase for sensitive PCR applications and high yields
  • Antibody-based Hot Start for fast polymerase activation

Applications

  • Hot-start PCR up to 3 kb
  • Amplification of low-copy-number targets
  • RT-PCR and TA cloning

Purchase Hot Start Taq DNA Polymerase, 5 U/µl

Availability: In stock Bulk requests please inquire at oem@biotechrabbit.com

Catalog # Size Package Content Price* Qty
BR0200102 500 U 100 µl Hot Start Taq DNA Polymerase
2 × 1.8 ml 5× PCR Reaction Buffer
1.5 ml 50 mM MgCl2
€80.00
BR0200103 2500 U 5 kits BR0200102 Hot Start Taq DNA Polymerase (500 U each)
€360.00

*Does not include applicable tax, shipping & handling, or other charges.

OR

biotechrabbit™ Hot Start Taq DNA Polymerase is a first-choice hot-start PCR enzyme for all demanding PCR applications. The enzyme ensures high product yields with low background and without primer–dimer formation and nonspecific priming.

The Hot Start Taq DNA Polymerase is inactive during reaction setup due to the bound antibody which is quickly released at elevated temperatures, ensuring the enzyme is active only during PCR. There is no need for prolonged heating or denaturation steps.

The optional use of 5× PCR Enhancer improves PCR results in many cases, including impure template or low template abundance.

 

Component

Composition

Hot Start Taq DNA Polymerase

Hot Start Taq DNA Polymerase, 5 U/µl, in storage buffer
containing 50% (v/v) glycerol

5× Reaction Buffer

Optimized PCR buffer without magnesium ions

50 mM MgCl2

50 mM MgCl2 in water

 

STORAGE

−20°C (until expiry date – see product label)

 

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 Hot Start Taq 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 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:

Final concentration of MgCl2 in a 50 µl reaction, mM

2.0

2.25

2.5

2.75

3.0

Volume of 50 mM MgCl2 solution to add, µl

2.0

2.25

2.5

2.75

3.0

 

Basic Protocol
  • Optionally, use the supplied 5× PCR Enhancer to increase the yield and to lower the background in more complicated PCR reactions (low amounts of template, impure or GC-rich template).
  • 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 Hot Start PCR Master Mix, 2× (cat. no. BR0200201)
  • 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.

Component

Volume

Final concentration

5× Reaction Buffer

10 µl

50 mM MgCl2

Variable (standard 1.5 µl)

1.5–3 mM

 

Higher than 2 mM MgCl2 might increase yield but reduce fidelity

5× PCR Enhancer (optional - BR1900201)

10 µl

10 mM dNTP Mix

1 µl

200 µM

Forward primer

Variable

0.2–1 µM

Reverse primer

Variable

0.2–1 µM

Template DNA

Variable

10 pg–1 μg

 

Use 0.01–1 ng for plasmid or phage DNA and 0.05–1 μg for genomic DNA

Hot Start Taq DNA Polymerase (5 U/µl)

0.4–0.5 µl

2–2.5 U

Nuclease free water

Variable

 

Total volume

50 µl

 

  • 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

Step

Temperature

Time

Cycles

Initial activation

95°C

2 min

1

Denaturation

95°C

30 s

25–35

Annealing*

(55-68°C)

15–30 s

25–35

 

*Recommended annealing temperature is 5°C below Tm of primers.
U
se gradient PCR to optimize the annealing temperature

Extension

72°C

30–60 s/kb

25–35

Final extension

72°C

5 min

1

 

To extend all incomplete PCR products

Storage in the cycler

4°C

Indefinitely

1

  • Add loading dye solution (see DNA Loading Dye, 6×, cat. no. BR0800301) to the reactions to analyze PCR products on a gel or store them at −20°C.
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