 |
|
 |
|
|
| Home >
Products >
Polymerases and Amplification >
Mesophilic DNA Polymerases >
Bsu DNA Polymerase, Large Fragment |
 | | | | Bsu DNA Polymerase, Large Fragment | | | |
|
|
|
 |
|
Prices are in US dollars and valid only for US orders.
|
Description:
Bsu DNA Polymerase I, Large Fragment retains the 5´→ 3´ polymerase activity of the Bacillus subtilis DNA polymerase I (1), but lacks the 5´→ 3´ exonuclease domain. This large fragment naturally lacks 3´→ 5´ exonuclease activity.
Source:
An E. coli strain that contains a genetic fusion of the Bacillus subtilis DNA polymerase I gene (starting from codon 297 thus lacking the 5´→ 3´ exonuclease domain), and the gene coding for maltose binding protein (MBP). The fusion protein is purified to near homogeneity and the MBP portion is cleaved off in vitro. The remaining DNA polymerase is purified free of MBP.
Applications:- Random primer labeling
- Second strand cDNA synthesis
- Single dA tailing
- Strand displacement DNA synthesis (2)
Reagents Supplied:
NEBuffer 2 (10X)
Enzyme Properties
Polymerase Properties | Thermophilic Polymerase Characteristics
3´ to 5´ Exonuclease: No
5´ to 3´ Exonuclease: No
Strand Displacement: Yes
Heat Inactivation:
75°C for 20 minutes
Specific Activity:
20,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 2
Supplemented with 33 μM dNTPs (not included)
Incubate at
37°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTPs into acid insoluble material in 30 minutes at 37°C.
Unit Assay Conditions:
1X NEBuffer 2, 33 µM dNTP including [3H]-dTTP and 70 µg/ml denatured herring sperm DNA. Incubate at 37°C
Concentration:
5,000 units/ml
Storage Temperature:
-20°C
Notes
Usage notes:- Bsu DNA Polymerase, Large Fragment is not suitable for generating blunt ends because it lacks the 3´→ 5´ exonuclease necessary to remove non-templated 3´ additions.
- Bsu DNA Polymerase, Large Fragment is also active in all NEB restriction enzyme reaction buffers when supplemented with dNTPs.
- Bsu DNA Polymerase, Large Fragment retains 50% activity at 25°C and is twice as active as Klenow Fragment (3´→ 5´ exo–) at this temperature.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified free of contaminating endonucleases and exonucleases.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of Bsu DNA Polymerase, Large Fragment with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (105 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of Bsu DNA Polymerase, Large Fragment with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
3´→ 5´ Exonuclease Activity::
Incubation of a 20 µl reaction containing 50 units of Bsu DNA Polymerase Large Fragment and a 10 nM solution of fluorescent 5´ -FAM labeled oligonucleotdie for 30 minutes 37°C yields no detectable 3´→ 5´ degradation as determined by high resolution capillary chromatography.
References
- Okazaki, T. et al. (1964) J. Biol. Chem., 239, 259-268.
- Piepenburg, O. et al. (2006) PLOS Biology, 4, 1115-1121.
Reagents Sold Separately
NEBuffer 2
Companion Products
Deoxynucleotide Solution Mix
Deoxynucleotide Solution Set
|
|
|
 |
|
 |
|
|
