Experimental design, in a 25μl system, using the same amount (0.1ng) of pCMV-Cre-EGFP as the template, using different amounts of this product or Bst DNA polymerase of Competitor, primer: 1.6µM FIP/BIP, 0.2µM F3/B3, 0.4µM Loop F/B, 1.4mM dNTP each, MgSO4 was added to 1X Bst Reaction Buffer (2mM MgSO4) until the final concentration reached 8mM, and incubated at 65ºC for 1 hour. Inactivation was heated at 80ºC for 20 minutes and then tested by 2.0% agarose gel electrophoresis.
As shown in the figure, this product has comparable enzyme activity compared with Competitor N's products.
Lane 1 Negative Control-1(negative control with no added enzyme only)
Lane 2 Negative Control-2(only negative controls without templates)
Lane 3 UA070061- Bst DNA Polymerase, Large fragment 8U
Lane 4 competing product N 8U
Bst DNA Polymerase, Large fragment
Bst DNA Polymerase, Large fragment
Product Details
Product Details
Product Specification
| Synonyms | DNA polymerase I |
| Expression System | E.coli |
| Molecular Weight | 67kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE |
| Tags & Cleavage sites | / |
| Tag | His Tag |
| Storage Buffer | 50 mM KCl、10 mM Tris-HCl、0.1 mM EDTA、1 mM DTT、0.1% Triton® X-100、50% Glycerol、pH 7.1 @ 25°C |
| Stability & Storage | Store at -25 ~ -15℃ for 2 years |
| Reference | 1. Nucleic Acids Research, 2000, 28(12):E63. |
Background
Bst DNA Polymerase, Large Fragment is the portion of the Bacillus stearothermophilus DNA Polymerase protein that contains the
5´ → 3´ polymerase activity, but lacks 5´ →3´ exonuclease activity. It can be use to isothermal amplification (LAMP), DNA sequencing through high GC regions and Rapid Sequencing from nanogram amounts of DNA template.
Components
50 mM KCl、10 mM Tris-HCl、0.1 mM EDTA、1 mM DTT、0.1% Triton® X-100、50% Glycerol、pH 7.1 @ 25°C
10* Reaction Buffer: 200 mM Tris-HCl、100 mM (NH4)2SO4、20mM MgSO4、100 mM KCl、1% Tween® 20、pH 8.8@25°C
Magnesium Sulfate (MgSO4) Solution：100mM MgSO4
Protocol
Incubate the following reaction at 65°C for 30–60 minutes
Component |
Final Concentration |
10X Isothermal Amplification Buffer II |
1X (contains 2 mM MgSO4) |
MgSO4 (100 mM) |
6 mM (8 mM total) |
dNTP Mix (10 mM) |
1.4 mM each |
FIP/BIP Primers (25X) |
1.6 µM |
F3/B3 Primers (25X) |
0.2 µM |
LoopF/B Primers (25X) |
0.4 µM |
Bst DNA Polymerase (8,000 U/ml) |
320 U/ml |
DNA or RNA Sample |
> 10 copies or more |
Nuclease-free Water |
to 25 µl |
Total Reaction Volume |
25 µl |
Guidelines
1.Bst DNA Polymerase does not exhibit 3´→ 5´ exonuclease activity.
2.100 µg/ml BSA or 0.1%Triton X-100 is required for long term storage.
3.Reaction temperatures above 70°C are not recommended.
4.Bst DNA Polymerase, Large Fragment cannot be used for thermal cycle sequencing or PCR
Unit Definition
Picture
Picture
Bioactivity
SDS-PAGE
2μg (R: reducing condition, N: non-reducing condition).
SEC-HPLC
95.5%
