|
Pfu
DNA polymerase is a thermostable enzyme isolated from
Pyrococcus furiousus. The enzyme replicates DNA at
75°C, catalyzing the polymerization of nucleotides into
duplex DNA in the 5'-3' direction. Pfu
DNA polymerase possesses 3'-5' exonuclease
(proofreading) activity. Base misinsertions that may
occur during polymerization are rapidly excised by the
proofreading activity of the polymerase.
Pfu
DNA polymerase is recommended for use in
PCR and primer extension reactions that require
high-fidelity synthesis.
Pfu
DNA polymerase-generated PCR fragments
are blunt-ended.
Reaction Buffer (10x) with MgSO4:
200 mM TrisHCl (pH 8.8 at 25°C), 100 mM
KCl,
100 mM (NH4)2SO4,
20 mM MgSO4, 1.0% Triton X-100
Storage conditions:
-20oC.
Procotol：
|
Step |
Temperature |
Time |
Cycle |
|
Initial denaturation |
94-95oC |
1-3 min |
1 |
|
Denaturation |
94-95oC |
0.5-2 |
25-35 |
|
Annealing |
37-70 |
0.5-2 |
25-35 |
|
Extension |
70-75 |
2min / 1kb |
25-35 |
|
Final extension |
70-75 |
5 |
1 |
Comparison of DNA polymerase and Pfu
DNA polymerase
Pfu
DNA polymerase
500U/100ul (5unit/ul)
|
Feature/characteristic |
DNA
polymerase |
Pfu
DNA polymerase |
|
5‘-3’ polymerase |
yes |
yes |
|
5‘-3’ exonuclease |
No |
yes |
|
dNTP incorporate rate/sec |
35 - 100 |
- |
|
Error rate |
2.4 x 10-5 |
0.4 x 10-5 |
|
Thermostability at 95oC |
40 min > 50% |
240 min > 70% |
|
longest amplify |
5 kb |
10 kb |
|
additional A tail |
yes |
No |
|
application |
PCR, RT-PCR |
High fidelity, long PCR |
|
