67kDa (Reducing)
Store at -25 ~ -15℃ for 2 years
1. Nucleic Acids Research, 2000, 28(12):E63.
2. Chemical Communications, 2014, 50(28):3747-3749.
3. Current Protocols in Molecular Biology, 2014(Suppl.105):15.14.1-15.14.14.
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.
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
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 |
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
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
2μg (R: reducing condition, N: non-reducing condition).
95.5%
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