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YourTaq™ Direct-Load Hot Start PCR Mix, 2×

Features

  • Exceptionally pure YourTaq Hot Start DNA Polymerase
  • Optimized Master Mix for increased yield of amplification and direct loading on the gel
  • Resistant to PCR inhibitors for direct PCR amplification
  • Excellent PCR specificity and sensitivity for a broad range of amplicons

Applications

  • Routine and demanding PCR amplification up to 3 kb
  • Suitable for amplification of low target copy number
  • TA cloning

Purchase YourTaq™ Direct-Load Hot Start PCR Mix, 2×

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

Catalog # Size Package Content Price* Qty
BR0202301 200 rxn of 50 µl 4 × 1.25 ml YourTaq Hot Start Direct-Load PCR Mix
€102.00
BR0202302 1000 rxn of 50 µl 20 × 1.25 ml YourTaq Hot Start Direct-Load PCR Mix
€404.00
BR0202303 4000 rxn of 50 µl 4 kits BR0202302 YourTaq Hot Start Direct-Load PCR Mix (1000 rxn each)
€1,422.00

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

OR

biotechrabbit™ YourTaq Direct-Load Hot Start PCR Mix is optimized for high yield of amplification of 0.1–3 kb DNA targets, even from low copy number. YourTaq Hot Start PCR Mix shows excellent PCR specificity and sensitivity for a broad range of amplicons. The mix is resistant to PCR inhibitors, such as blood (up to 20%), Ethanol or humic acid enabling direct PCR amplification without any DNA extraction.

The 2× YourTaq Direct-Load Hot Start PCR Mix contains pure biotechrabbit YourTaq Hot Start DNA Polymerase, extremely high-quality dNTPs, two dyes (blue and yellow) that separate during electrophoresis, allowing migration progress to be monitored, and sufficient buffer density for direct loading onto agarose gels. In addition, the mix is suitable for amplification of GC-rich templates (up to 70%) pairing with 5× PCR Enhancer (BR1900201).

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

 

Component

Composition

YourTaq Direct-Load Hot Start PCR Mix

Optimized 2× YourTaq Hot Start PCR Master Mix containing electrophoresis tracking dyes (yellow and blue) and density reagent for direct gel loading

 

STORAGE

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

 

Quality Control
Functional assay

Human genomic DNA was amplified using the 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.

Component

Volume

Final concentration

YourTaq Direct-Load Hot Start PCR Mix, 2×

25 µl

5× PCR Enhancer (optional - BR1900201)

10 µl

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

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 2°C above Tm of primers.
Use 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

  • Directly load the reactions on a gel to analyze PCR products or store them at −20°C.
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