Pfu DNA Pol is a superior thermostable DNA Pol isolated from an E.coli strain carrying the “pol” gene from Pyrococcus furiosus with 5’ →3’ polymerase activity and 3’ → 5’ exonuclease proofreading activity, i.e. it catalyzes DNA synthesis with very low error rate (about twelve times more accurate than Taq Pol). However, Pfu DNA Pol is slower than Taq Pol and typically requires about 1-2 min per cycle to amplify 1Kb of DNA.Using Pfu DNA Pol in PCR reactions also results in blunt-ended amplification products. Pfu DNA Pol is hence superior for techniques that require high fidelity DNA synthesis, but can also be used in conjunction with Taq Pol to obtain the fidelity of Pfu with the speed of Taq Pol activity.
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Pfu DNA Polymerase
cat. no.
amount
note
STS-P250
STS-P1000
250 units
10X Standard Buffer
10X Standard Buffer
STS-Pw250
STS-Pw1000
1000 units
250 units
10X Standard Buffer w/o MgCl2
10X Standard Buffer w/o MgCl2
cat. no.
amount
note
STSn-P250
STSn-P1000
1000 units
250 units
10X Standard Buffer + dNTPs
10X Standard Buffer + dNTPs
STSn-Pw250
STSn-Pw1000
1000 units
250 units
10X Standard Buffer w/o MgCl2 + dNTPs
10X Standard Buffer w/o MgCl2 + dNTPs
add nucleotides box
FOR RESEARCH USE ONLY
UNIT DEFINITION
One unit is defined as the amount of enzyme required to incorporate 10 nanomoles of dNTPs into acid-insolubile material in 30 min at 74°C.
SHIPPING
Shipped in green ice.
STORAGE
Store at -20C°
SHELF LIFE
12 months
FORM
Liquid
CONCENTRATION
2U/ul
component
STS-P250
STS-Pw250
STPn-T250
STSn-Pw250
Pfu Polymerase
Standard Buffer
MgCl2
dNTPs
250 units / 125ul
1ml 10X Buffer with MgCl2
1ml 10X Buffer w/o MgCl2
1ml 10X Buffer with MgCl2
1ml 10X Buffer w/o MgCl2
250 units / 125ul
250 units / 125ul
250 units / 125ul
-
-
100ul / 10mM each
100ul / 10mM each
-
500ul / 50mM
-
500ul / 50mM
component
STS-T1000
STS-Tw1000
STSn-T1000
STSn-Tw1000
Pfu Polymerase
Standard Buffer
MgCl2
dNTPs
1000 units / 500ul
2ml 10X Buffer with MgCl2
2ml 10X Buffer w/o MgCl2
2ml 10X Buffer with MgCl2
2ml 10X Buffer w/o MgCl2
1000 units / 500ul
1000 units / 500ul
1000 units / 500ul
-
-
400ul / 10mM each
400ul / 10mM each
-
500ul / 50mM
-
500ul / 50mM
Assay Set-Up:
Before starting, vortex all components thoroughly to ensure homogeneity.
Prepare a premix for the number of assays you need according to the following protocol:
component
Standard Buffer
dNTPs
Pfu Polymerase
primers
1000 units
DNA Template
MG Water
stock conc.
10X
10mM each
2U/ul
1ug/ul each
-
-
final conc.
1X
200uM
0.5U/ul
50ng/ul each
-
-
50ul reaction
5.0ul
1.0ul
1.0ul
3.4ul each
10-100ng
up to 50ul
> Pfu DNA Pol
Cycling conditions:
Spin down the tubes/plate briefly to remove bubbles and place them into the cycler.
denaturation
denaturation (1)
annealing (2)
extension
95°C
95°C
50-68°C
72°C
5min
45 sec -1min
45 sec -1min
1 -2 min
5 min
1)The annealing temperature depends on the melting temperature of the primers used.
2)The elongation time depends on the length of the fragments to be amplified. A time
of 1 min/kb is recommended.
high fidelity
PCR and Cloning
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Laboratory
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molecular
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