Description
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 specific DNA polymerase-mediated reactions by relaxing DNA secondary structure and enhancing enzyme processivity. This enzyme 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.
Storage temperature: –25°C to –15°C Molecular weight: 18,975 Daltons Test Units tested Specification Purity n/a >99% Single-stranded exonuclease 25 µg <1% released Double-stranded exonuclease 25 µg <1% released Double-stranded endonuclease 25 µg No conversion E. coli DNA contamination n/a 0.7 µg inhibits PCR
The recombinant enzyme protein is produced by a recombinant E. coli strain carrying the E. coli gene for the single-stranded DNA binding protein.
Usage Instructions The E. coli SSB is a single-stranded DNA binding protein (SSB) that can be used to stabilize single-stranded DNA regions and enhance DNA polymerase fidelity and processivity in PCR (1,5). The protein can also be used to minimize deletion mutagenesis artifact during Taq DNA polymerase mediated PCR (4), improve the yield of DNA amplification, and enable sequencing of problematic DNA templates, for example, regions with strong secondary structures (5). As a starting point in SSB-mediated PCR reactions, add E. coli SSB protein at a concentration range between 20–320 ng/μL per 50 µL reaction to identify the optimal concentration. References: Meyer, R.R. and Laine, P.S. (1990) Microb. Rev., p342-380. Krauss, G. et al. (1981) Biochemistry., 20, 5346-52. Weiner, J.H. et al. (1975) J. Biol. Chem., 250, 1972-80. Chou, Q. (1992) Nuc. Acids. Res., 20(16), 4371. Rapley, R. (1994) Mol. Biotechnol., 2, 295-298. Quality Control Protein concentration is determined by OD 280 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 band's mass 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 the amount 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 needed to inhibit PCR amplification of 5 ng target DNA following 25 cycles of PCR.
