Bst DNA Polymerase，Large Fragment
The large segment Bst DNA polymerase is a part of the Bacillus stearothermophilus DNA polymerase, derived from the E. coli strain. By using gene recombination technology, it is expressed in Escherichia coli and purified and separated multiple times. This enzyme has 5 '→ 3' DNA polymerase activity, but does not have 5 '→ 3' exonuclease activity. This product can be used for DNA sequencing, DNA labeling, and isothermal DNA amplification.
Product features:
1. Strong chain displacement activity.
2. High sensitivity.
3. The optimal activity temperature is 65 ℃, with constant temperature amplification and no need for a thermal cycling instrument.
Product Usage:
1. DNA sequencing rich in GC sequences;
2. Rapid sequencing of trace (nanogram) DNA templates;
3. Random primer DNA labeling;
4. Double stranded DNA 5 'protruding end patch labeling;
5. It can be used for isothermal DNA amplification, such as loop mediated isothermal amplification (LAMP), whole genome amplification (WGA), helicase constant temperature gene amplification (HDA), etc.
Product composition:
Component 800U
Bst DNA Polymerase, Large Fragment 100 μ L
ten × Reaction buffer 1mL × two
Storage condition: -20 ℃
Example of reaction:
The large fragment of Bst DNA polymerase was used in LAMP experiment. M13 ssDNA was used as template, F3/B3 and FIP/BIP were used as primers, and after reaction at 65 ℃ for 1 hour, agarose gel electrophoresis was performed.
Swimming lane M: 1kb DNA Ladder;
Lane 1: LAMP amplification product.
Quality assurance:
After multiple column purification, only a clear and single target band was visible in SDS-PAGE gel detection. The qPCR method detected no residual Escherichia coli DNA and no contamination of nucleic acid endonucleases or exonucleases.
Activity definition:
The amount of enzyme required to incorporate 10 nmol of dNTPs into acid insoluble precipitates within 30 minutes at 65 ℃ is defined as one active unit.
ten × Reaction buffer:
200mM Tris HCl, 100mM (NH4) 2SO4500mM KCl, 20mM MgSO4, 1% Tween 20, pH8.8.
Storage solution:
50mM KCl, 10mM Tris HCl (pH7.5), 1mM DTT, 0.1mM EDTA, 0.1% Triton X-100, and 50% Glycerol.
Reaction conditions: 1 × Reaction buffer, 65 ℃ warm bath.
Thermal deactivation: 80 ℃, 10 minutes
Precautions:
1. Bst DNA polymerase does not exhibit 3 '→ 5' exonuclease activity;
2. Long term storage requires adding 100 μ G/ml BSA or 0.1% Triton X-100;
3. It is recommended that the reaction temperature should not exceed 70 ℃;
4. Bst DNA polymerase cannot be used for thermal cycling sequencing or PCR.
