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Pfu PCR Master Mix, 2x

Features

  • Pure Pfu DNA Polymerase and highest quality dNTPs in a 2x Pfu PCR Master Mix formulation
  • Accurate PCR for demanding applications,  about ten times higher accuracy than that of Taq DNA Polymerase
  • High value for a fair price

Applications

  • High Throughput High Fidelity PCR
  • Generation of PCR products for blunt cloning

Purchase Pfu PCR Master Mix, 2x

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

Catalog # Size Package Content Price* Qty
BR0300201 100 react. of 50 µl 2x 1.25 ml Pfu PCR Master Mix; 0.25 ml 5x PCR Helper
€107.00
BR0300202 500 react. of 50 µl 10x 1.25 ml Pfu PCR Master Mix; 1.2 ml 5x PCR Helper
€450.00

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

OR

The biotechrabbit Pfu PCR Master Mix is a perfect choice for a fast high fidelity PCR reaction set up without the need for calculation, multiple pipetting steps and buffer optimization. It is designed for routine high throughput high fidelity PCR amplifications of up to 4kb size DNA targets.

The 2x Pfu PCR Master Mix contains Pfu DNA polymerase, highest quality dNTPs and optimal PCR reaction buffer components, thus only the template and PCR primers have to be added. The final reaction volume should be reached with the pure PCR grade water.

For the most demanding applications the supplied PCR additive 5x PCR Helper can be used. It is not necessary to use for every reaction, but in many cases it helps to achieve even better results with templates having high GC content and complex structures.

The biotechrabbit Pfu DNA Polymerase is a highly purified recombinant thermostable proofreading DNA polymerase. The enzyme is isolated from E. coli carrying a plasmid with the cloned Pyrococcus furiosus DNA polymerase gene.

Pfu DNA Polymerase has about ten times higher accuracy compared to Taq DNA Polymerase.

The enzyme catalyzes template-dependent nucleotides polymerization in the 5'-3' direction, additionally it exhibits 3'-5' exonuclease (proofreading) activity, what allows the correction of nucleotide incorporation errors.

The enzyme has no 5'-3' exonuclease activity and no detectable reverse transcriptase activity.

The use of dUTP and dITP is not recommended with Pfu, because Pfu binding to DNA templates with uracil and hypoxanthine stalls DNA synthesis.

Pfu DNA Polymerase produces blunt ended PCR products suitable for blunt cloning.

Component
Composition

Pfu PCR Master Mix, 2x

Proprietary composition, contains all necessary PCR reaction components

5x PCR Helper

Proprietary composition

Storage

Store at -20°C for 12 months. Avoid multiple freeze thaw cycles by preparing aliquots.

Quality Control

Components tested:

Endodeoxyribonuclease Assay

No detectable conversion of covalently closed circular DNA to nicked DNA was observed after incubation of 15 units of Pfu DNA Polymerase with 0.5 µg of pUC19 plasmid DNA 10 µl of 1x Reaction Buffer containing 1.75 mM MgCl2 for 2 hours at 72°C.

Exodeoxyribonuclease Assay

No detectable degradation (smearing) of fragments was observed after incubation of 5 units of Pfu DNA Polymerase with 0.5 µg of pUC19 plasmid DNA (digested with Hpa II) in 10 µl of 1x Reaction Buffer containing 1.75 mM MgCl2 for 2 hours at 72°C.

Ribonuclease Assay

No detectable degradation of 28S/18S RNA bands was observed after incubation of 5 units of Pfu DNA Polymerase with 1 µg of total RNA (from rat liver) in 10 µl of 1x Reaction Buffer containing 1.75 mM MgCl2 for 2 hours at 72°C.

Master Mix tested:

Functional Assay

0.1 ng of lambda DNA was amplified using specific primers to produce a distinct 4k bp band.

Prevention of PCR reaction contamination

PCR is a very sensitive DNA amplification reaction; therefore care should be taken to eliminate the possibility of contamination with any foreign DNA templates.

  • Use separate clean areas for preparation of the samples, reaction mixture and for cycling.
  • Wear fresh gloves.
  • Use sterile tubes and pipette tips with aerosol filters for PCR reaction set up.
  • Use DNA and Nuclease free water and other reagents.
  • With every PCR reaction set up perform a contamination control reaction without template DNA.
Typical PCR reaction set up

The typical PCR protocol without optimization is usually working for uncomplicated routine PCR amplifications with average GC content, pure templates and up to 3 kb size targets. But the best conditions for a certain primer –template PCR reaction vary and are typically optimizes by:

  • Choosing the optimal amounts of template and primers
  • Trying the supplied 5x PCR Helper solution – an additive for a better PCR results.
  • Optimizing cycling conditions.
Basic Protocol with or without the 5x PCR Helper

The use of PCR Helper is not necessary in general, but helps in the amplification of DNA with high GC content and complex structures. The 5x PCR Helper can be added to the reaction mixture at the final concentration of 0.5x – 2x according to the guidelines below.

  • Thaw on ice and mix very well all reagents
  • Assemble and keep all reactions on ice.
  • Pipet the master mix into the thin walled 0.2ml  PCR tubes. Add separately the component which is differing, typically template or primers.

Component

Volume (in µl)

PCR Helper not used

PCR Helper at 0.5x concentration

PCR Helper at 1x concentration

PCR Helper at 1.5x concentration

PCR Helper at 2x concentration

5x PCR Helper

0

5

10

15

20

Pfu PCR Master Mix, 2x

25 µl

Forward-Primer

0.2-1 µM final concentration

Reverse-Primer

0.2-1 µM final concentration

Template DNA

1-5ng of plasmid or phage DNA; 50-500ng of genomic DNA; Too much template increase the background, too low amounts of template reduce the PCR accuracy.

Nuclease free water

add up to 50

  • Mix and collect the drops by centrifuging briefly.
  • Overlay with 50 μl of mineral or silicone oil if this is required for your PCR machine.
  • Place into the PCR cycler.
Cycling Program

Step

Temperature (in °C)

Time

Cycles

Initial activation

95

2 min

1

Denaturation

95

30 sec

25– 35

Annealing

5°C below Tmelting of primers

45 sec

25 – 35

Extension

72

2 min/kb

25 – 35

Final Extension

72

5 min

1

Storage in the cycler

4°C

until the reactions are taken out.

1

  • Add the loading dye solution and analyze PCR reactions on the gel or store them at -20oC up to they are analyzed.