BstDNA Polymerase 1.0

$290
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SKU:
HH7001
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Bst DNA Polymerase is the portion of the Bacillus stearothermophilus DNA Polymerase protein that contains the 5´→3´polymerase activity but lacks 5´→3´exonuclease activity. This product uses E. coli expression and is suitable for isothermal amplification.

Store Buffer:

10 mM Tris-HCl (pH 7.1),50 mM KCl,0.1 mM EDTA,1 mM DTT,0.1% Triton X-100,50% Glycerin.

Application

  1. Isothermal amplification.
  2. DNA sequencing with high GC content.
  3. Rapid Sequencing from nanograms of DNA template.

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.

Quality Control

  • Endonuclease residue detection: The 50 μL reaction system added 500 U enzyme, 1 μg of PUC19 DNA, and incubated at 37℃ for 4h without degradation signal.
  • Exonuclease residue detection: After incubation at 37℃ for 16 h, the electrophoretic bands of DNA did not change after adding 500 U enzyme and 1 μg Lambda-HindIII DNA into the reaction system.
  • RNase residue detection RNase: 8 U enzyme and 40 ng RNA were incubated at 37℃ for 16 h, and the electrophoresis band of RNA did not change.
  • E. coli DNA residue detection: The remaining nucleic acid in 120 U of this enzyme was detected by TaqMan qPCR for E. coli gDNA, and the E. coli genome residue was less than 10 copies.

Experiment Process

  1. Take out 10×Bst Buffer, thaw on ice, vortex for 10 seconds before use. Mix well and collect by centrifugation briefly.
  2. In addition to the template DNA, the remaining components are configured in the order shown in the table below.
Component Volume Final Con
10 ×Bst Buffer 2.5 μL 1 ×(2 mM Mg2+ )
MgSO4 (100 mM) 1.5 μL 6 mM (total 8 mM)
dNTP Mix (10 mM) 3.5 μL 1.4 mM
FIP/BIP Primers (25×) 1 μL 1.6 μM
F3/B3 Primers (25×) 1 μL 0.2 μM
LoopF/LoopB Primers 1 μL 0.4 μM
Bst DNA Polymerase(8 U/μL) 1 μL 320 U/mL
Template DNA x μL >10 copies
Nuclease-free Water up to 25 μL
     

3. Mix by pipetting, and centrifuge briefly to collect. 4. Constant temperature incubation at 60°C- 65°C for 1 hour.

  1. The Bst Buffer can be dissolved at room temperature. After dissolution, it should be stored in an ice box or ice bath. After using, it should be stored at -20℃ immediately.
  2. For your safety and health, please wear lab coat and disposable gloves.