Description
Product: Pfu DNA Polymerase (with HighPure dNTP mix)
Cat. No.: P***2
Item No.: P***2
Storage conditions: Store at **0℃. Concentration: 5U/µl
Product composition:
Pfu DNA Polymerase **0U
*0×Pfu Buffer (with MgSO4) 1ml
Pure dNTP mix (*0 mM Each) 0.2ml
Product description:
Pfu DNA Polymerase is isolated and purified from Escherichia coli
cloned with Pyrococcus furiosis DNA Polymerase gene. Pfu DNA
Polymerase has 5′*3′ DNA polymerase activity and 3′*5′ exonuclease
activity, which can correct base mismatches generated during DNA
amplification. Pfu enzyme has the lowest error rate among all the
high-temperature resistant DNA Polymerases discovered so far. Its
PCR product is blunt-ended and can be directly cloned with a
blunt-ended vector
Activity unit:
1 unit (U) Pfu DNA Polymerase activity is defined as the amount of
enzyme required to incorporate *0nmol of deoxynucleotides into
acid-insoluble substances using activated salmon sperm DNA as a
template primer at *4°C within *0 minutes.
Quality control:
SDS-PAGE detection purity is greater than *9%, and no exogenous
nuclease activity is detected; PCR method detection shows no host
residual DNA, and can effectively amplify single-copy genes in the
human genome; no obvious activity changes after storage at room
temperature for one week.
Enzyme storage buffer:
*0mM Tris-HCl (pH 8.2), 0.1mM EDTA, 1mM DTT, Stabilizers, *0%
glycerol.
*0×Pfu Buffer (containing Mg2+):
**0mM Tris-HCl (pH8.8), **0mM KCl, **0mM(NH4)2SO4, *0mM MgSO4,
other ingredients.
Scope of application:
Used for high-fidelity amplification of DNA, such as gene
expression cloning, gene site-directed mutagenesis, intracellular
gene point mutation analysis (SNP) and blunt-end
complementation.
Notes:
(1) Pfu enzyme has 3′*5′ exonuclease activity, so the extension
speed of Pfu enzyme amplification is lower than that of Taq enzyme.
The corresponding extension time should be set according to the
length of the amplified product. It is recommended that the
extension speed of Pfu enzyme is 1 kb per minute if the amplified
fragment is less than 4 kb; the extension speed is 0.5 kb per
minute if the amplified fragment is greater than 4 kb. At the same
time, the 3′*5′ exonuclease activity of Pfu enzyme may degrade
primers, so dNTPs should be added first, then Pfu enzyme should be
added to the reaction system, and PCR reaction should be carried
out immediately.
(2) When using Pfu enzyme for amplification, the purity of the
primers is required to be higher, the primer length is greater than
*8 bases, the Tm is between ****0℃, and the primer concentration
is between 0.**0.5 μM, which is slightly higher than Taq
enzyme.
(3) Pfu enzyme has better thermal stability than Taq enzyme. For
templates with high GC content, the denaturation temperature can be
increased to *8℃ without affecting the activity of Pfu
enzyme.
(4) High-fidelity PFU has very high requirements for dNTP purity,
so it is recommended to use the ultra-pure dNTP mix that comes with
this enzyme.
Recommended PCR conditions: (taking *0μl reaction system as an
example)
Template <0.5 μg
Forward Primer (*0 μM) 1 μl
Reverse Primer (*0 μM) 1 μl
*0×Buffer (With MgSO4) 5 μl
SuperPure dNTP Mixture (*0mM each) 1 μl
Pfu DNA polymerase (5U/μl) 0.5μl
dH2O up to *0 μl
PCR reaction cycle settings: (Using plasmid as template
amplification, 1kb/min is generally sufficient.)
*4°C: **5 min
*4°C: *0 sec
****0°C: *0 sec
*2°C: 2 min/1 kb
*2°C: ***0 min
*0cycles
