Quality control analysis
Unit activity is measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X RNase H reaction buffer and added to 50 µs reactions containing 3H-labeled poly(rA), poly (dT) DNA and 1X RNase H Buffer. Reactions were incubated 20 minutes at 37°C, placed on ice and release of TCA soluble counts was analyzed.
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 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 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 is determined in a 50 µl reaction containing 0.5 µg plasmid DNA and 10 µl enzyme solution that was 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.