Prime RNase Inhibitor

Description
Prime RNase Inhibitor is a protein of non-human origin that binds non-covalently and inhibits the same types of ribonuclease as HPRI, including RNases A, B and C. Prime RNase Inhibitor can be used in cDNA synthesis, in in vitro transcription using the SP6, T7 and HeLa cell extract systems and in in vitro translations using rabbit reticulocyte lysates and wheat germ extracts.

Additionally, Prime RNase Inhibitor prevented RNA degradation in cell extracts prepared under non-denaturing conditions1, and was utilized in first strand synthesis prior to PCR2 and in the RT-PCR of RNA isolated from single cells.

Prime RNase Inhibitor was used in gel retardation assays, the isolation of an RNA binding protein by oligonucleotide affinity purification, nuclear run-on transcription assays as well as in vitro transcription in S. cerevisiae whole cell extracts, and in polysome distribution analysis at +4°C.

Prime RNase Inhibitor is most active in the absence of DTT. A slight inhibition of activity is measured in the presence of an increasing concentration of DTT. In the presence of 20 mM DTT, the activity of Prime RNase Inhibitor is approximately 90% of its activity in the absence of DTT.

Storage Buffer

2.0 mM KH2PO4, 8.0 mM Na2HPO4, 3.0 mM KCl, 150 mM NaCl, pH 7.4 and 50% Glycerol

Concentration: 30 units/µl

About the Units: 30 units of the Prime RNase Inhibitor are required per 30 µl sample.

Storage at -20°C in constant temperature freezer

Applications

In vitro transcription
In vitro translation
First and second strand cDNA synthesis
Preparation of RNA and mRNA
Reverse transcription-PCR

Product features

Inhibits greater than 90% of RNase activity where Human Placental RNase Inhibitors (HPRI) inhibits only 50%
Stable under a broad range of pH, DTT concentrations, and temperatures
Inhibits RNases A, B, and C, just like HPRI, and does not interfere with RNases T1, T2, H, U1, U2, or CL3

Prime RNase Inhibitor1 is an extremely potent RNase inhibitor intended for use in experiments where RNase contamination may exist. Prime RNase Inhibitor is supplied at a concentration of 30 Units per µl in 2.0 mM KH2PO4, 8.0 mM Na2HPO4, 3.0 mM KCl,
150 mM NaCl, pH 7.4, and 50% Glycerol.

Amount of RNase Inhibitor

Potency of Prime RNase Inhibitor compared to HPRI

Store at -20°C in a non-frost-free freezer.
Prime RNase Inhibitor also may be stored at 2-8°C for up to 12 months with no apparent decrease in activity.
The addition of DTT is not necessary or recommended for storage.
Do not store at -70°C or below.
Prime RNase Inhibitor is fully active for at least one year when properly stored unopened as described.

Working dilution
Use of 1 Unit Prime RNase Inhibitor per 1 µl of reaction volume is recommended. Further dilution of Prime RNase Inhibitor may not provide an adequate degree of RNase inhibition. Whenever possible, Prime RNase Inhibitor should be added to
reactions prior to addition or synthesis of RNA. If severe RNase contamination is suspected, it is recommended that Prime RNase Inhibitor be pre-incubated with all reaction components, excluding RNA, for 1/2 hour at room temperature.

Prime RNase Inhibitor is a protein ribonuclease inhibitor that non-covalently binds and inactivates a variety of RNase A-type ribonucleases (see table below).

Inhibition	No Inhibition
RNase A	RNase T1 RNase U1
RNase B	RNase T2 RNase U2
RNase C	RNase H RNase CL3

Prime RNase Inhibitor is most active in the absence of DTT. A slight inhibition of activity is measured in the presence of increasing concentrations of DTT.2 In the presence of 20 mM DTT, the activity of Prime RNase Inhibitor is approximately 90% of its activity in the absence of DTT. The addition of DTT is not necessary or recommended for storage. Prime RNase Inhibitor will be inactivated when exposed to denaturing conditions.

For applications requiring incubation at temperature over 37°C, please consult the table below.

Temperature	Stability
50°C	< 60 minutes
60°C	< 30 minutes
65°C	< 20 minutes
70°C	< 3 minutes

Unit definition
One unit of Prime RNase Inhibitor will inhibit greater than 90% of the activity of 5 ng of pancreatic RNase A (pRNase A). This is determined in a 30 µl reaction containing 40 µg yeast RNA, 150 ng pRNase A, 25 mM Hepes-KOH, pH 7.6, 25 mM Tris-Cl, pH 7.5, 10 mM MgCl2, 50 mM KCl, 1 mM spermidine, 1 mM EDTA, 5 mM DTT, and Prime RNase Inhibitor. Reactions are incubated at 37°C for 2 hours and analyzed by 1% agarose gel electrophoresis.

Quality assurance
Each lot of Prime RNase Inhibitor contains no detectable RNase or DNase activity as determined by overnight incubation of 30 Units of Prime RNase Inhibitor with yeast RNA and λ Hind III fragments, respectively, and subsequent agarose gel analysis. Sterility is tested by plating 2 µl onto a DYT-agar plate and incubating at 37°C for 14 to 16 hours. No growth is evident on the plate. 15 units of Prime RNase Inhibitor is tested in a first strand synthesis reaction and compared to a reaction without Prime RNase Inhibitor to ensure that Prime RNase Inhibitor does not inhibit first strand synthesis.

Article	Order no.
Prime RNase Inhibitor (7,500 U)	0032005357
Prime RNase Inhibitor (15,000 U)	0032005403