Quality control analysis
Unit activity is determined using 1.1-fold serial dilution method. Dilutions of enzyme were made in 1X RNase Inhibitor Reaction Buffer and added to 1000 µl reactions containing 1mM cytidine 2’,3’-cyclic monophosphate, 1 µg RNase A in a 1X reaction buffer containing 100 mM Tris-Acetate, 1 mM EDTA, pH 6.5. Absorbance at 286 nm was observed at 20 second intervals during a 5-minute reaction.
Protein concentration (OD280) 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 protein of interest band in the diluted sample.
Single-stranded exonuclease activity 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 16S rDNA contamination is evaluated using 5 µl replicate samples of enzyme solution 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.
Non-specific RNase contamination is assessed using the RNase Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.