Description
RecA, which functions in DNA recombination and DNA repair (1, 2, 3), binds to single stranded DNA, resulting in the polymerization of RecA into a nucleoprotein complex. This complex aligns with complementary-double stranded DNA, resulting in RecA catalysis of DNA strand exchange. RecA DNA binding is stimulated by ATP hydrolysis or non-hydrolyzable ATP analogs. The RecA–ATP–single-stranded DNA complex also functions as a coprotease factor in the proteolytic cleavage of LexA, UmuD and certain bacteriophage proteins. RecA complexed with site-specific oligonucleotides has been used to target and specifically cleave large DNA fragments (4). Supplied in: 10 mM Tris-Cl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.5 at 25°C. Supplied with: 10X RecA Reaction Buffer (B9260): 700 mM Tris·Cl, 100 mM MgCl2 and 50 mM DTT; pH 7.6 at 25°C.
Enzyme properties Storage temperature: –25°C to –15°C Molecular weight: 37,973 Daltons Test Amount tested Specification Purity N/A >95% Single-stranded exonuclease 2 µg <5.0% released Double-stranded exonuclease 2 µg <1.0% released Double-stranded endonuclease 2 µg No conversion E. coli DNA contamination 5 µg <10 copies
Source of recombinant enzyme protein The protein is produced by an E. coli strain overexpressing the E. coli recA gene from a plasmid. Unit definition: Sold by mass of pure protein determined at OD 280 (A 280 = 0.516 at 1 mg/m, 1cm).
Quality control analysis 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 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. E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.
