Bsu DNA Polymerase (Large Fragment) is derived from Bacillus subtilis. It is obtained by truncating the N-terminal 296 amino acids of Bacillus subtilis DNA Polymerase I. This enzyme retains the 5'→3' polymerase activity of Bacillus subtilis DNA Polymerase I, while lacking both 5'→3' and 3'→5' exonuclease activities. It exhibits an optimal reaction temperature of 37 °C and possesses strong strand displacement activity, making it suitable for DNA isothermal amplification and second-strand cDNA synthesis.
Quality Control
1. Protein Purity
Determined by SDS-PAGE; purity ≥ 95%.
2. Endonuclease Activity Assay
5 μg Bsu DNA Polymerase was incubated with 200 ng supercoiled plasmid DNA at 37 °C for 4 h. Agarose gel electrophoresis showed less than 10% of plasmid DNA was converted into nicked or linear form.
3. Non-specific Nuclease Activity Assay
5 μg Bsu DNA Polymerase was incubated with 15 ng double-stranded DNA fragment at 37 °C for 16 h. No alteration of dsDNA substrate was observed by agarose gel electrophoresis.
4. RNase Activity Assay
5 μg Bsu DNA Polymerase was incubated with 500 ng total RNA at 37 °C for 1 h. Agarose gel electrophoresis confirmed over 90% of RNA remained intact.
5. Host Genomic DNA Residue
Quantitative real-time PCR with E. coli 16S rDNA-specific primers and probes was used to detect 5 μg Bsu DNA Polymerase; residual E. coli genomic DNA was less than 10 copies.
Inactivation Condition
Incubation at 75 °C for 20 min completely inactivates the protein.
Precautions：
- Due to the absence of 3'→5' exonuclease activity, Bsu DNA Polymerase (Large Fragment) cannot remove unpaired 3' overhangs, and thus is not suitable for blunt-end generation.
- Bsu DNA Polymerase (Large Fragment) retains 50% of its activity at 25 °C, which is twice that of Klenow Fragment (3'→5' exo⁻).
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