Bsu DNA Polymerase, Large Fragment

Bsu DNA Polymerase, Large Fragment is expressed by a recombinant E. coli strain cloned from the Bacillus Subtilis DNA polymerase I gene and obtained by multi-step purification. This large fragment retains the 5 ´ → 3 ´ polymerase activity of the Bacillus subtilis DNA polymerase I, but lacks the 5´→3´ exonuclease domain (starting from codon 297 thus lacking the 5 ´ → 3 ´ exonuclease domain). This large fragment naturally lacks 3 ´ → 5 ´ exonuclease activity.

Storage Temperature

Store at -20℃, valid for one year (Avoid repeated freeze-thaw cycles).

Storage Buffer

25 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol and 50% glycerol.

Unit Definition

One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTPs into acid insoluble material in 30 minutes at 37°C.

Heat Inactivation

75°C for 20 minutes.

Quality Control Assays

Exonuclease Activity: 5 U of Bsu DNA Polymerase, Large Fragment with 1 μg λ-Hind III digest DNA at 37℃ for 4 hours yields no degradation as determined by agarose gel electrophoresis.

Endonuclease Activity: 5 U of Bsu DNA Polymerase, Large Fragment with 1 μg λDNA at 37 ℃ for 4 hours yields no degradation as determined by agarose gel electrophoresis.

Nicking Activity: 5 U of Bsu DNA Polymerase, Large Fragment with 1 μg pBR322 at 37℃ for 16 hours yields no degradation as determined by agarose gel electrophoresis.

RNase Activity: 5 U of Bsu DNA Polymerase, Large Fragment with 1.6 μg MS2 RNA for 16 hours at 37 ℃ yields no degradation as determined by agarose gel electrophoresis.

E.coli DNA: 5 U of Bsu DNA Polymerase, Large Fragment is screened for the presence of E. coli genomic DNA using TaqMan qPCR with primers specific for the E. coli 16S rRNA locus.The E. coli genomic DNA contamination is E. coli genome.

Reaction Conditions

1X Bsu Reaction Buffer： 10 mM Tris-HCl (pH 7.9 @ 25°C) 50 mM NaCl 10 mM MgCl2 1 mM DTT

Notes On Use

• Bsu DNA Polymerase, Large Fragment is not suitable for generating blunt ends because it lacks the 3 ´ → 5 ´ exonuclease necessary to remove non-templated 3´ additions.

• Bsu DNA Polymerase, Large Fragment retains

• Random primer labeling
• Second strand cDNA synthesis
• Single dA tailing
• Strand displacement DNA synthesis