 |
|
 |
|
|
| Home >
Products >
Polymerases >
Thermophilic DNA Polymerases >
Bst DNA Polymerase, Large Fragment |
 | | | | Bst DNA Polymerase, Large Fragment | | | |
|
|
|
|
 |
|
Prices are in US dollars and valid only for US orders.
|
- Isolated from a recombinant source
- Specific Activity: 120,000 units/mg
- Sequencing through problematic secondary structures
- Supplied with 10X Reaction Buffer
Description:
Bst DNA Polymerase Large Fragment is the portion of the Bacillus stearothermophilus DNA Polymerase protein that contains the 5´ → 3´ polymerase activity, but lacks the 5´ →3´ exonuclease domain.
Source:
Bst Polymerase Large Fragment is prepared from an E. coli strain containing a genetic fusion of the Bacillus stearothermophilus DNA Polymerase gene, lacking the 5´ → 3´ exonuclease domain, and the gene coding for E. coli maltose binding protein (MBP). The fusion protein is purified to near homogeneity, and the MBP portion of the fusion is cleaved off in vitro. The remaining polymerase is purified free of MBP (1).
Applications:- DNA sequencing through high GC regions (2,3)
- Rapid Sequencing from nanogram amounts of DNA template (4)
Reagents Supplied:
ThermoPol Reaction Buffer (10X)
Enzyme Properties
Polymerase Properties | Thermophilic Polymerase Characteristics
3´ to 5´ Exonuclease: No
5´ to 3´ Exonuclease: No
Strand Displacement: Yes
Heat Inactivation:
80°C for 20 minutes
Molecular Weight:
Theoretical: 67,000 daltons
Specific Activity:
120,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X ThermoPol Reaction Buffer
Incubate at
65°C.
1X ThermoPol Reaction Buffer:
20 mM Tris-HCl
10 mM (NH4)2SO4
10 mM KCl
2 mM MgSO4
0.1 % Triton X-100
pH 8.8 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 10 nmoles of dNTP into acid insoluble material in a total reaction volume of 50 μl in 30 minutes at 65°C in 50 mM KCI, 20 mM Tris-HCI (pH 8.8), 10 mM MgCl2, 30 nM M13mp18 SS DNA, 70 nM M13 sequencing primer (-47) 24 mer, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 100 µM dTTP, 3H dTTP and 100 µg/ml BSA.
Concentration:
8,000 units/ml and 120,000 units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
0.1% Triton X-100
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- Bst DNA Polymerase does not exhibit 3´→ 5´ exonuclease activity.
- 100 µg/ml BSA or 0.1%Triton X-100 is required for long term storage.
- Reaction temperatures above 70°C are not recommended.
- Bst DNA Polymerase cannot be used for thermal cycle sequencing.
FAQs
- Can Bst DNA Polymerase be used in other NEBuffers?
- Can Bst DNA Polymerase be used to blunt DNA?
- Can Bst DNA Polymerase be used to fill in 3' overhangs?
- Can Bst DNA Polymerase be used to remove 5' overhangs?
- Can Bst DNA Polymerase be heat inactivated?
- Are NEB DNA Polymerases supplied with dNTPs?
- What are the main causes of reaction failure using Bst DNA Polymerase?
- Does Bst DNA Polymerase have an active 3'→5' proofreading exonuclease?
- Can Bst DNA Polymerase be used for thermal cycle sequencing?
- Can Bst DNA Polymerase initiate at a nick in the DNA?
- Can Bst DNA Polymerase be used in labeling reactions and partial fill in reactions?
- Can Bst DNA Polymerase be diluted?
- When should Bst DNA Polymerase be the enzyme of choice?
- Can Bst DNA Polymerase be used at temperatures other than 65°C?
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.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA
and 500 units of Bst DNA Polymerase, Large Fragment incubated for 16 hours at 65ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 500 units of Bst DNA Polymerase, Large Fragment with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 65ºC
released < 0.05% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 500 units of Bst DNA Polymerase, Large Fragment with 1 μg of
ΦX174 RF I DNA for 4 hours at 65ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
References
- Kong, H., Aliotta, J. and Pelletier, J.J., New England Biolabs, unpublished observations.
- Griffin, H. and Griffin, A. (1994) PCR Technology, 228-229.
- McClary, J. et al. (1991) J. DNA Sequencing and Mapping, 1, 173-180.
- Mead, D.A. et al. (1991) Biotechniques, 11, 76-87.
Reagents Sold Separately
ThermoPol Reaction Buffer
Companion Products
BSA
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,814,506
|
|
|
 |
|
|
