Psp DNA Polymerase(Pfu)
Code  BN0050-0500
Classification  PCR試劑 & 1-Step Kit
Size  500u
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Psp DNA polymerase (DNA-tested)

Description:
 
Pfp DNA polymerase, isolated from the hyperthermophilic archae bacteria Pyrococcus furiosus is a thermostable Polymerase of approximately 92 kDa. The enzyme replicates DNA at 75°C, catalyzing the polymerization of nucleotides into duplex DNA in the 5´-->3´ direction in the presence of magnesium (prefers MgSO4). Unlike Taq DNA polymerase, Pfp DNA polymerase possesses 3' to 5' exonuclease proofreading activity that enables the polymerase to correct nucleotide-misincorporation errors. This means that Pfp DNA polymerase-generated PCR fragments will exhibits the lowest error rate of any thermostable DNA Polymerase, a 12-fold increase in fidelity of DNA synthesis compared with Taq DNA polymerase. Pfp DNA polymerase is recommended for use in PCR and primer extension reactions that require high-fidelity synthesis. Pfp DNA polymerase generated PCR fragments are blunt-ended, which can be used directly for blunt end ligation. Pfp DNA Polymerase exhibits lower than that of Taq DNA Polymerase extension rate (0.5kb/min), so 2min extension time is recommended for every 1 kb to be amplified.
   
  Pfp DNA Polymerase prefers MgSO4 to MgCl2.
   
  Error rate (x10-5): 0.2
   
 
For 50μl volume start from 1.25 – 2.5 units. Meanwhile, for minute amount of template the amount of enzyme should be reduced substantially. For optimal performance use dilutions 1/2, 1/10, 1/50 of the enzyme supplied and use 1 μl of each dilution for the reaction.
   
  One minute extension time is sufficient for PCR fragments up to 0.5-1 kb
   
Purity test
 
Psp DNA polymerase is free from endonucleases activities, no traces of bacterial DNA was found in PCR-reactions with primers normally designed for amplification of superconservative region of rDNA
   
Storrage buffer
  50mM TrisHCl, pH8,2; 0,1 mM EDTA; 1mM DTT; 0,1% Nonidet P40; 0,1% Tween 20; 50% glycerol
   
Concentration
  5000 units/ml
 
Supplied Buffers:
   
DILUTION BUFFER:
  TrisHCl, pH8,2; 0,1 mM EDTA; 1mM DTT; 0,1% Nonidet P40; 0,1% Tween 20; 50% glycerol
   
INCOMPLETE:
  (10X) 200 mM Tris-HCl (pH 8,8), 100 mM KCl, 100 mM (NH4)2SO4, 1,0 % Triton X-100, 1 mg/ml nuclease-free BSA
   
COMPLETE:
  (10X) 200 mM Tris-HCl (pH 8,8), 100 mM KCl, 100 mM (NH4)2SO4 , 20 mM MgSO4, 1,0 % Triton X-100, 1 mg/ml nuclease-free BSA
One tube MgSO4 (100 mM)
   
Storage conditions
  Storage temperature is -20℃.
   
Protocol (example for minute amount of genomic DNA)
  Be careful with the amount of the enzyme!
  The influence of Psp DNA polymerase amount on the performance of Psp-directed PCR

PCR was performed in 25μl on Eppendorf Master cycler with the following program:

94℃ – 2 min, initial denaturation step

30 cycles:
94℃ – 10sec
55℃ – 20sec
72℃ – 1 min 30sec

72℃ – 5 min – final filling-in

Template – Genomic DNA of rodent Microtus arvalis (10ng/μl)
Primers:
M7 – 5’-TATGTGCCTTTCCTATAAGC (20pmol/μl)
T10 – 5’AAGCAGGTATCCATTACC (20pmol/μl)
10pmoles of each primer was used in PCR reaction

Amplicon – 720bp fragment of Xist gene

10xreaction buffer:
100mM ammonia sulfate
200mM TrisHCl, pH 8,8
100mM KCl
20mM MgSO4
1% Triton X-100
1mg/ml BSA
  Reaction mixture:
 
dNTPs mixture (8mM of each) – 0.5 μl
10x buffer 2,5 μl
primer M7 (20μM) 0,5 μl
primer T10 (20μM) 0,5 μl
DNA 10ng/μl 1 μl
Psp DNA –polymerase x μl
Water up to 25μl
 
Table: amountμl of Psp DNA polymerase in the reaction.
   
 
The optimal amount of Psp DNA polymerase is 0,016-0,062 μl of the enzyme, it corresponds to 0,08-0,3 units of the enzyme per reaction. The increase of enzyme concentrations results in unspecific products formation (0,625-1,3u) and in the complete disappearance of the product at 2,5u.