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2X Hot-Start PCR Master Mix


  • Highest PCR sensitivity without prolonged reactivation
  • Optimized PCR Master Mix for minimal hands-on and fast setup
  • Exceptionally pure Hot Start Taq DNA Polymerase and highest quality dNTPs


  • High-specificity and high-throughput hot-start PCR up to 3 kb
  • Amplification of low-copy-number targets
  • TA cloning

Purchase 2X Hot-Start PCR Master Mix

Availability: In stock Bulk requests please inquire at

Catalog # Size Package Content Price* Qty
BR0200205 200 reactions of 50 µl 4 × 1.25 ml Hot-Start PCR Master Mix
BR0200206 1000 reactions of 50 µl 20 × 1.25 ml Hot-Start PCR Master Mix
BR0200207 4000 reactions of 50 µl 4 kits BR0200206 Hot-Start PCR Master Mix (1000 rxn each)

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


biotechrabbit™ Hot Start PCR Master Mix is a perfect choice for a fast reaction setup that reduces the time required for calculation and pipetting and eliminates the need for buffer optimization. It is designed for low-background, high-throughput PCR of 0.2–3 kb DNA targets.

The 2x Hot Start PCR Master Mix contains pure biotechrabbit Hot Start Taq DNA Polymerase, extremely high-quality dNTPs and optimized PCR buffer; thus, only template, PCR primers and PCR-grade water are added.

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 hot start minimizes primer–dimers and mispriming.

Info: Recommended annealing temperature is 2°C above Tm of primers or use gradient PCR to optimize the annealing temperature.




Hot Start PCR Master Mix

Optimized 2× Hot Start PCR Master Mix



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


Quality Control
Functional assay

Human genomic DNA was amplified using the Hot Start PCR Master Mix and specific primers to produce a distinct band.


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 the amplification of long targets, 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
  • Using a PCR Enhancer (i.e. BR1900201) for low amounts of template, impure or GC-rich templates
  • Optimizing cycling conditions
Basic Protocol
  • The Master Mix is designed to be used without any optimization as it has all necessary reaction components in optimal amounts for successful PCR.
  • 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.
  • Keep all reagents and reactions on ice.
  • 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.



Final concentration

Hot Start PCR Master Mix, 2×

25 µl

5× PCR Enhancer (optional - BR1900201)

10 µl

Forward primer


0.2–1 µM

Reverse primer


0.2–1 µM

Template DNA


10 pg–1 μg


Use 0.01–1 ng for plasmid or phage DNA and 0.1–1 μg for genomic DNA

Nuclease free water



Total volume

50 µl


  • Mix and centrifuge briefly to collect the liquid in the bottom of the tube.
  • Place in the PCR cycler.
Cycling Program





Initial activation


2 min




30 s




15–30 s



*Recommended annealing temperature is 2°C above Tm of primers,
or use gradient PCR to optimize the annealing temperature



30–60 s/kb


Final extension


5 min



To extend all incomplete PCR products

Storage in the cycler




  • 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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