Download T4 DNA Polymerase

For general laboratory use.
FOR IN VITRO USE ONLY.
T4 DNA Polymerase
From T4 plasmid pTL43W infected Escherichia coli 71–18
Deoxynucleoside-triphosphate: DNA deoxynucleotidyl-transferase (DNA-directed), EC 2.7.7.7
Cat. No. 11 004 786 001
Cat. No. 11 004 794 001
100 U
500 U
Product overview
Pack content
Vial
Product
description
Enzyme
properties
Content
T4 DNA
polymerase
(1 U/l)
• 100 l (100 U pack size)
• 500 l (500 U pack size)
Enzyme storage buffer; 10 mM TrisHCl, 100 mM NaCl, 2.5 mM MgCl2,
0.5 mM EDTA, 2 mM dithioerythritol,
50% glycerol (v/v), pH 8.0 (4 °C)
Incubation
buffer, 5 conc.
• 1 ml ( 100 U pack size)
• 1 ml (500 U pack size)
Buffer composition: 250 mM Tris-HCl,
75 mM (NH4)SO4, 35 mM MgCl2,
0.5 mM EDTA, 50 mM 2-mercaptoethanol, 0.1 mg/ml bovine serum
albumin (BSA), pH 8.8 (25°C)
For polymerization reactions, supplement the buffer with dATP*, dCTP*,
dGTP* and dTTP*, 33 mol/l each
(final concentration). This buffer is
optimal for the polymerase and exonuclease activities of T4 DNA
polymerase
T4 DNA polymerase is a DNA dependent DNA polymerase that catalyzes the polymerization of deoxynucleoside-5´-triphosphates to the hydroxyl termini of
recessive ends. Blunt ended DNA cannot serve as template for the polymerization reaction. For polymerization T4 DNA polymerase requires DNA with 5´protruding ends and a high concentration of dNTP’s.
The enzyme carries an extremely active 3´→5´-exonuclease that shows a strong specificity for ssDNA (1)
and lacks a 5´→3´-exonuclease activity. Therefore
nicked duplex DNA cannot serve simultaneously as
template and primer for polymerization. The addition of
T4 gene 32 protein facilitates strand displacement and
therefore allows T4 DNA polymerase to replicate the
nicked duplex (2,3).
Note: Low levels of dNTP’s should not be used for
polymerization reaction because once the dNTP’s
are exhausted, the exonuclease activity will degrade
the DNA.
Volume activity ≥ 1 U/l of T4 DNA polymerase is the
amount of enzyme activity that incorporates 10 nmol dNTP into acid-precipitable DNA products in 30 min at
37°C( 4).
For lot-specific values see data label.
Specific
activity
≥ 5 U/g according to Gouliand and
Bradford (4,5).
For lot-specific values see data label
Purity
≥ 10 g of T4 DNA polymerase
migrate as a single band in SDSpolyacrylamide gel electrophoresis
according to Laemmli (6).
Mass weight
single polypeptide of 114 000
Optimal pH
8 9 (at pH 7.5 and 9.7 approx. 50%
activity is found)
Optimal Mg2+ approx. 6 mM
0904.11006835 5
Version Sept. 2004
Store at 15 to 25° C
Storage and
stability
The undiluted enzyme solution is stable at -15 to -25°C
until the control date printed on the label.
Application
T4 DNA polymerase is used to label 3´-termini of DNA.
Extensive labeling is achieved by the replacement
reaction, in which the 3´-exonuclease activity of the
enzyme first digests dsDNA to produce molecules with
recessed 3´-termini (7). On subsequent addition of
labeled dNTPs, the polymerase activity of T4 DNA polymerase then extends the 3´-ends along the length of
the template. Exonuclease III from E. coli can be used
to create partially single-stranded dsDNA for subsequent polymerization reactions (8). Molecules labeled
to high specific activity are used chiefly as hybridization probes. They have two advantages over probes
prepared by nick translation: they lack artificial
hairpin structures and they can easily be converted
into strand-specific probes by cleavage with
suitable restriction endonucleases (9). In combination
with T4 gene 32 protein T4 DNA polymerase is used
for gap-filling in site-directed mutagenesis
experiments (10).
Quality control
See data label for lot-specific values.
Test buffer
50 mM Tris-HCl, 7 mM MgCl2, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, 15 mM (NH4)2SO4, 0.1 mg/ml
BSA, pH approx. 7.5 (at 37° C).
Absence of
endonucleases
1. 1 g DNA is incubated with T4 DNA polymerase
for 4 h at 37° C in 50 l test buffer. The number of
enzyme units which show no degradation of DNA
is stated under “Endo 1”.
2. 1 g Eco RI/Hind III fragments of DNA is
incubated with T4 DNA polymerase for 4 h at 37° C
in 50 l test buffer. The number of enzyme units
which show no alteration of the banding
pattern is stated under “Endo 2”.
Absence of ssspecific DNases
1 g ss M13mp9 DNA are incubated with T4 DNA
polymerase for 4 h at 37° C in 50 l test buffer.
The number of enzyme units that show no degradation
of ss M13mp9 DNA is stated under “ss-Act”.
Absence of
nicking activity
1 g pBR322 DNA is incubated with T4 DNA polymerase for 4 h at 37° C in 50 l test buffer. The number
of enzyme units which show no relaxing of supercoiled
structure is stated unter “Nick. Act.”
Absence of
RNases
4 g MS2 RNA are incubated with T4 DNA polymerase
for 4 h at 37° C in 50 l test buffer. The number of
enzyme units which produce no change in the
appearance of MS2 RNA is stated under “RNase”.
References
1
2
3
4
5
6
7
Huang, W. M. & Lekman, I. R. (1972) J. Biol. Chem. 247, 3139.
Alberts, B. & Frey, L. (1970) Nature 227, 1313.
Nossal, N. G. (1974) J. Biol. Chem. 249, 5668.
Goulian, M. et al. (1968) J. Biol. Chem. 243, 627.
Bradford, M. (1976) Anal. Biochem. 72, 248.
Laemmli, U. K. (1970) Nature 227, 680.
Maniatis, T. et al. (1989) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor
Laboratory, New York.
8 Richardson, C. C. & Kornberg, A. (1964) J. Biol. Chem. 244, 2996.
9 Deen, K. D. et al. (1983) Anal. Biochem. 135, 456.
10 Craik, C. S. et al. (1985) Science 228, 291.
Ordering Information
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Product
Pack size
Cat. No.
100 µg
500 µg
10 972 983 001
10 972 991 001
DIG-Nick Translation
Mix
60 µl
11 745 816 001
Biotin-Nick Translation Mix
160 µl
11 745 824 001
Nick Translation Mix
200 µl
11 745 808 001
T4 Gene 32 Protein
Nick Translation Kit
1 kit
10 976 776 001
(50 radioactive
reactions)
DNA Polymerase I
500 units
1000 units
10 104 485 001
10 104 493 001
DNA Polymerase I,
endonuclease-free
250 units
1000 units
10 642 711 001
10 642 72 0001
DNase I, RNase-free
10000 units
10 776 785 001
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