RecA functions in DNA recombination and DNA repair (1,2,3). RecA binds to single stranded DNA, resulting in the polymerization of RecA into a nucleoprotein complex. This complex can align 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 can function as a coprotease factor in the proteolytic cleavage of LexA, UmuD and certain bacteriophage proteins. RecA complexed with site-specific oligonucleotides have been used to target and specifically cleave large DNA fragments (4).
Source of Protein
A recombinant E. coli strain overexpressing E. coli recA from a plasmid.
10 mM Tris-HCl
1 mM DTT
0.1 mM EDTA
pH 7.5 @ 25°C
B9260 (10X RecA Reaction Buffer)
10X RecA Reaction Buffer (B9260):
700 mM Tris-HCl
100 mM MgCl2
50 mM DTT
pH 7.6 @ 25°C
Sold by mass of pure protein determined at
OD280 (A280 = 0.516 at 1 mg/mL, 1cm).
Protein Concentration (OD280) Measurement
A 2.0 µL sample of enzyme was analyzed at OD280 using a Nanodrop ND-2000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209), and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 20,340 and molecular weight of 37,973 Daltons.
SDS-Page (Physical Purity Assessment) and Specifications
2.0 µL of enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µL of a 1:100 dilution of the sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 95% purity of the concentrated sample.
Single-Stranded Exonuclease Activity
A 50 µL reaction containing 10,000 cpm of a radiolabeled single-stranded DNA substrate and 10µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 5% release of TCA-soluble counts.
Double-Stranded Exonuclease Activity
A 50 µL reaction containing 5,000 cpm of a radiolabeled double-stranded DNA substrate and 10µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 1% release of TCA-soluble counts.
Double-Stranded Endonuclease Activity
A 50 µL reaction containing 0.5 µg of pBR322 DNA and 10µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.
E.coli 16S rDNA Contamination Test
Replicate 5µL samples of enzyme solution were 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. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.
|Unit Size||1.5 mg|
|SDS||Available on request|
|Storage Temperature||-15 to -25°C|
|SS Exonuclease||2 µg <5.0% released|
|DS Exonuclease||2 µg <1.0% released|
|DS Endonuclease||2 µg = no conversion|
|E.coli DNA Contamination||5 µg <10 copies|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. MSDS sheets relevant to this product are available upon request.
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