• High fidelity PCR

• Site-directed mutagenesis

• Generation of PCR products for Blunt end cloning

HiPhi¢â Plus DNA Polymerase, originated from an extreme thermophile, is ideal for high-fidelity PCR amplification. Its unique buffer system reduces primer-dimer formation and non-specific amplification. Due to its intrinsic 3¡¯ to 5¡¯ exonuclease (proof-reading) activity, this DNA polymerase shows high-fidelity and produces blunt ended PCR products HiPhi¢â Plus DNA Polymerase is an engineered enzyme showing much higher productivity and processivity than other proof-reading thermostable enzymes (e.g. Pfu DNA polymerase, Vent DNA polymerase etc.) without compromise to the fidelity. For the maximum performance of PCR reactions high-quality dNTP mixture is supplied. The addition of 5x Q Plus Buffer in the reaction mixture is a simple way to optimize the difficult targets of PCR.

 

 

* Chemical Hot Start DNA Polymerase with 3' to 5' exonuclease activity

* Reduced primer-dimer and non-specific amplification

* Broad range annealing temperature

    

PCR Reaction Conditions
¡¡	25 ul volume	50 ul volume
Template	X ul	X ul
10X Reaction Buffer	2.5 ul	5 ul
dNTP Mix ( each 10 mM )	0.5 ul	1 ul
Forward Primer (10 pmoles/¥ìl)	1 ul	2 ul
Reverse Primer (10 pmoles/¥ìl)	1 ul	2 ul
5x Q Plus Buffer(optional)	0 ~ 10 ul	0 ~ 20 ul
HiPhi¢â Plus DNA Polymerase	1.25 units	1.25 units
Distilled water	up to 25 ul	up to 50 ul

   

¡Ø 5x Q Plus Buffer is an additive altering the binding behavior of primer and template and can help the amplification that do not work well under standard PCR condition. Especially, 5x Q Plus Buffer can be used for the amplification of problematic template, such as high G+C content and repeat sequence regions. 5x Q Plus Buffer uses as adding into PCR reaction mixture from 0.5x to 2x.

 

 

Human genomic DNA	10 ng – 100 ng
Bacterial genomic DNA	5 ng – 50 ng
Purified plasmid or phage DNA	1 ng – 5 ng

  

 

Perform 25-40 cycles of PCR amplification as follows
Initial Denaturation	95¡É	2 minutes
25-40 cycles	95¡É 55 -68 ¡É 72¡É	20 seconds
		40 seconds
		2 minutes/kb
Final Extension	72¡É	5 minutes

50% Glycerol, 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, 0.1% Tween 20, 0.1% Nonidet P-40, 1 mM PMSF

 

The HiPhi¢â Plus DNA polymerase is shipped on Dry/Blue Ice. All kit components should be stored at -20¡É upon receipt. Excessive freeze/thawing is not recommended. When stored under optimum conditions, the reagents are stable for a minimum of 6 months from date of purchase.

 

HiPhi¢â Plus DNA polymerase was passed from quality control assay for contamination of bacterial host DNA using sequence-specific primer set from host bacterial genomic DNA.

 

Harmful if swallowed. Irritating to eyes, respiratory system and skin. Please refer to the material safety data sheet for further information.

 

1u is defined as amount of enzyme that required to catalyze the incorporation of 10 nmoles of dNTP into acid-insoluble material in 30 minutes at 72¡ÆC.

 

• Recombinant HiPhi¢â Plus DNA polymerase is the enzyme of choice for most PCR applications.

• The number of PCR cycles depends on the amount of template DNA in the reaction mix and on the expected yield of the PCR product. 25 -35 cycles are usually sufficient for the majority PCR reaction. Low amounts of starting template may require 40 cycles.