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1. lt gt MCLAB Molecular Cloning Laboratories User Manual Version 3 0 Revision Date 01 09 2014 Product name Pfu DNA Polymerase Cat AD 200 AD 205 AD 210 AD OEM Description Pfu DNA Polymerase is a highly thermostable DNA polymerase from the hyperthermophilic archaeum Pyro coccus furiosus The enzyme catalyzes the template dependent polymerization of nucleotides into duplex DNA in the 5 gt 3 direction Pfu DNA Polymerase also exhibits 3 gt 5 exonuclease proofreading activity that enables the polymerase to correct nucleotide incorporation errors It has no 5 gt 3 exonuclease activity The main difference between Pfu and alternative enzymes is Pfu s superior thermostability and proofreading properties Unlike Taq DNA polymerase Pfu DNA polymerase also possesses 3 gt 5 exonuclease proofreading activity resulting in PCR fragments with fewer errors than Tag generated PCR inserts Pfu DNA polymerase is efficient for techniques that require high fidelity DNA synthesis but can also be used in conjunction with Taq polymerase to obtain the fidelity of Pfu with the speed of Taq polymerase activity Supplied with 10x Pfu Reaction Buffer with dNTP Supplied in 20 mM Tris HCl pH 8 0 40 mM NaCl 2 mM Sodium Phosphate 0 1 mM EDTA 1 mM DTT Stabilizers 50 v v glycerol Unit Definition One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmol of dNTPs into a2. cid insoluble material in 30 minutes at 74 C under standard DNA polymerase assay conditions Protocol 1 Add template DNA 10 pg 1 ng for plasmid and 0 1 1 ug for genomic DNA and both forward and re verse primers 200 nM of each final concentration to the PCR tube 2 Add 10x Pfu Reaction Buffer to 1 10 of final volume add nuclease free water to bring the total volume to final volume Component Volume Volume Template DNA 10 50 ng 10 50 ng 5 Primer 5 uM 1 uL 2 uL 3 Primer 5 uM 1 uL 2 uL 1 650 872 0245 www mclab com 1 10x Pfu Reaction Buffer 2 5 uL 5 uL with dNTP Pfu DNA Polymerase 1 uL 2 uL dH O To 25 uL To 50 uL FINAL VOLUME 25 uL 50 uL 3 Mix the PCR mixture thoroughly and spin down briefly 4 Place the PCR tubes into the PCR machine and start the PCR reaction 5 Load 2 uL or 5 uL reaction mixture directly on agarose gel to check the result after PCR reaction Load the rest on agarose gel if the amplified fragment need to be gel purified for downstream experiment 2 lt MCLAB
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