Bst DNA Polymerase, Full Length is the full length polymerase from Bacillus stearothermophilus. It has 5´ → 3´ polymerase and double-strand specific 5´ → 3´ exonuclease activity, but lacks 3´ → 5´ exonuclease activity. It can be used to isothermal DNA amplification and Primer extension.
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、100 mM KCl、20mM MgSO4、0.1% 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
Bst DNA Polymerase does not exhibit 3´→ 5´ exonuclease activity.
Reaction temperatures above 70°C are not recommended.
Cannot be used for thermal cycle sequencing or PCR.
Unit Definition
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 65°C
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