Source

E. coli strain expressing a recombinant clone that carries Ribonuclease Inhibitor gene from human placenta.

Concentration

40 U/μl.

Unit Definition

One unit is defined as the amount of Recombinant Ribonuclease Inhibitor required inhibit the activity of 5ng of ribonuclease A by 50%.

Activity is measured by the inhibition of hydrolysis of cytidine 2’3’-cyclic monophosphate by ribonuclease A．

Storage Buffer

20mM HEPES-KOH(PH7.6), 50mM KCl, 8mM DTT, 50%(v/v) glycerol

Storage

Store at -20°C

Quality Control

Absence of Endonuclease
1μg of Lambda DNA is incubated with 200 units of Ribonuclease Inhibitor for 16 hours at 37°C.
Following incubation, Lambda DNA is visualized as intact on an ethidium bromide-stained agarose gel to verify the absence of visible Endonuclease.

Absence of Nickase
1μg of Type I supercoiled pBR322 is incubated with 200 units of Ribonuclease Inhibitor for 4 hours at 37°C.
Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting.

Absence of Exonuclease
1μg of Lambda DNA/HindIII Markers is incubated with 200 units of Ribonuclease Inhibitor for 16 hours at 37°C.
Following incubation, Lambda DNA/HindIII Markers is separated by 1% agarose gel and stained with ethidium bromide. Markers remain as intact bands without smearing.

Absence of RNase and latent RNase
To test for the presence of RNase activity, 1ug of RNA is incubated with 200 units of Ribonuclease Inhibitor for 4 hours at 37°C.
Following incubation, the RNA is visualized as intact band on an ethidium bromide-stained agarose gel to verify the absence of visible RNase.
To test for the presence of latent RNase activity, 200units of Ribonuclease Inhibitor is heat-denatured at 70°C for 15minutes and incubated with 1ug of RNA for 4 hours at 37°C.
Following incubation, the RNA is visualized as intact band on an ethidium bromide-stained agarose gel to verify the absence of visible RNase.

Physical Purity

The purity is ≥95% as judged by SDS-polyacrylamide gel with Coomassie blue staining.

Usage Note

Ribonuclease Inhibitor is active over a broad pH range (pH5.5-9). The standard RT and in vitro transcription uses Ribonuclease Inhibitor at a final concentration of 1u/ul.

Order

RI039

RNase Inhibitor, Human Placenta

recombinant Human Placenta Ribonuclease Inhibitor, 40U/μl