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PallĀ® 47 mm, In-line Filter Holders

Item #: ASFILTHOLD47 
Pfu DNA Polymerase

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Pfu DNA PolymeraseĀ 
Pfu DNA polymerase is a heat stable DNA polymerase which has 5' 3' DNA polymerase and 3' 5' exonuclease (proofreading) activities. Pfu DNA polymerase retains the high fidelity, sensitivity and processivity with an error rate six-fold lower than Taq DNA polymerase, and significantly lower than the error rates of most other proofreading enzymes or DNA polymerase mixtures (1). This product is supplied with the unique Intact Genomics 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM, and 5X Magic Enhancer that enables efficient amplification of GC rich templates up to 84%. The physical purity of this enzyme is 98% as assessed by SDS-PAGE with Coomassie® blue staining.

Quality ControlĀ 
Quality control is performed following the production of each new lot of product to ensure that it meets the quality standards and specifications designated for the product.Ā Each lot is repeatedly compared side-by-side with leading competitors to ensure our products outperform the competitor before product launching.Ā 

Product Source
E. coli strain expressing aĀ PfuĀ DNA Polymerase gene fromĀ Pyrococcus furiosus.

Contents & Storage

  1. PfuĀ DNA Polymerase
  2. 10x PCR Buffer with Mg²+
  3. 5x Magic Enhancer
  4. 10 mM dNTP (Cat. # 3312d, 3314d only)

Store all contents at -20 °C.

Storage BufferĀ 
50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 °C.

10X PCR Buffer with Mg2+Ā 
100 mM Tris-HCl pH 9.0, 15 mM MgCl, 100 mM KCl, 80 mM (NH4)2SO4, 0.5% Igepal CA 630

Unit DefinitionĀ 
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTP into acid-insoluble form in 30 minutes at 72 °C.

Protocol
1.Ā Thaw 10x PCR Buffer, dNTP mix, primer solutions, 5x Magic Enhancer (if required) and mix thoroughly before use.Ā 
2.Ā Prepare a reaction mix according to the following table: The reaction mix typically contains all the components needed for PCR except the template DNA.

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PCR reaction set up:

Template DNA

xµl (0.01-0.5µMg)

10x PCR Buffer

10.0µl

dNTP (10 mM)

2.0µl

Forward Primer

xµl (0.1- 0.5µMM)

Reverse Primer

xµl (0.1- 0.5µMM)

5x Magic Enhancer (optional)

20µl

PfuĀ DNA Polymerase (5 U/µl)

0.5µl

H2O up to

100.0µl

3.Ā Mix the reaction mixture thoroughly.
4. Add template DNA to the individual PCR tubes containing the reaction mixture.Ā 
5.Ā Program the thermal cycler according to the manufacturerâs instructions. A typical PCR cycling program is outlined in the following table.

PCR cycling conditions:

Steps

Temp.

Time

CyclesĀ 

Initial denaturation

95 °C

3-5 min

1

Denaturation

94 °C

30-60 secĀ 

Ā Ā 

25-35

Annealing

52-66 °C

30-60 secĀ 

Extension

72-74 °C

1-2 minĀ 

Final extension

72-74 °C

10 minĀ 

1

HoldĀ 

4-12 °C

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6.Ā Place the PCR tubes in the thermal cycler and start the cycling program.

PNPCRREAG

Magnesium
Mg
Dye
No Dye
Enzymes
Intact Genomics
Pkg
Indiv
Type
Proofreading
 
Item#:
ASPRIMADNAPOL
 
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