Bst 3.0 DNA Polymerase is an in silico designed homologue of Bacillus stearothermophilus DNA Polymerase I, Large Fragment engineered for improved isothermal amplification performance and increased reverse transcription activity. Bst 3.0 DNA Polymerase contains 5´→3´ DNA polymerase activity with either DNA or RNA templates and strong strand displacement activity, but lacks 5´→3´ and 3´→5´ exonuclease activity. Bst 3.0 DNA Polymerase demonstrates robust performance even in high concentrations of amplification inhibitors and features significantly increased reverse transcriptase activity compared to Bst DNA Polymerase.



Fast, single-enzyme RT-LAMP can be performed using Bst 3.0 
RT-LAMP was performed using indicated DNA polymerase and Jurkat total RNA and primers for two genes (ACTB, left; HMBS2, right). Fastest results were observed with a 2-enzyme system, Bst 2.0 and WarmStart RTx, but robust amplification was also observed using Bst 3.0 without additional RT. Bst LF, Bst 2.0 and competitor enzymes showed highly variable performance, with slow threshold times or reaction failure on one of the two targets.

Product Source

An E. coli strain that carries the engineered Bst 3.0 gene.

Reagents Supplied

The following reagents are supplied with this product:

Applications

Isothermal amplification (LAMP, RT-LAMP)

Applications requiring strand-displacement DNA synthesis

Reverse transcription at elevated temperature (to 72°C)

Amplification in the presence of impure samples or amplification inhibitors

Unit Definition

One unit is defined as the amount of enzyme that will incorporate 25 nmol of dNTP into acid insoluble material in 30 minutes at 65°C.

Reaction Conditions

1X Isothermal Amplification Buffer II 
Incubate at 65°C

1X Isothermal Amplification Buffer II :
20 mM Tris-HCl
10 mM (NH₄)₂SO₄
150 mM KCl
2 mM MgSO₄
0.1% Tween® 20
pH 8.8 @ 25°C

Storage Temperature

-20°C

Storage Conditions

100 mM KCl
10 mM Tris-HCl
0.1 mM EDTA
1 mM DTT
0.1% Triton® X-100
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

80°C for 5 min

Unit Assay Conditions

50 mM KCl, 20 mM Tris- HCl (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 including [³H]-dTTP and 100 μg/ml BSA. 

Companion Products

• WarmStart® RTx Reverse Transcriptase
• Magnesium Sulfate (MgSO₄) Solution
• Isothermal Amplification Buffer II Pack
• Deoxynucleotide (dNTP) Solution Set
• Deoxynucleotide (dNTP) Solution Mix

1.    Bst 3.0 DNA Polymerase does not exhibit 3´→ 5´ exonuclease activity.

2.    Reaction temperatures above 72°C are not recommended.

3.    Bst 3.0 DNA Polymerase cannot be used for PCR or reactions requiring thermal denaturation.