E. coli Single-Stranded DNA Binding Protein

• Binds single-stranded DNA
• Relaxes secondary DNA structure and enhances processivity in certain DNA polymerase-mediated reactions

E. coli Single-Stranded DNA Binding Protein preferentially binds single-stranded DNA, forming a tetramer of four identical 18.9 kDa subunits, which protects 8–16 nucleotides. The protein does not bind well to double-stranded DNA. In nature, the protein participates in DNA replication, recombination and repair functions. In vitro, E. coli Single-Stranded DNA Binding Protein has been found to stimulate certain DNA polymerase-mediated reactions by relaxing DNA secondary structure and enhancing enzyme processivity.

This enzme is supplied in 50 mM Tris-HCl, 200 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.5 at 25°C.

SDS is available upon request.

Storage temperature: –25°C to –15°C

Molecular weight: 18,975 Daltons

Source of recombinant enzyme protein

The protein is produced by a recombinant E. coli strain carrying the E.coli gene for the single-stranded DNA binding protien.

Quality control analysis

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Single-stranded DNA binding ability was confirmed in a PCR-inhibition assay by adding decreasing amounts of E. coli Single-Stranded SDNA Binding Protein to a series of PCR amplifications containing target DNA, 200 µM dNTPs, 1X PCR buffer and Taq DNA Polymerase. Reactions were incubated in a thermal cycler and subjected to 25 PCR cycles. Samples were resolved using agarose gel electrophoresis and amount of required to block 100% accumulation of PCR product was recorded. Acceptance criteria for assay: 0.70 µg E. coli Single-Stranded SDNA Binding Protein is required to inhibit PCR amplification of 5 ng target DNA following 25 cycles of PCR.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Primer sequesteration