Murine RNase Inhibitor
Properties
Quality level: 100
Test: > 95% (SDS-PAGE)
Mole weight: ~ 50 kDa
Packaging
- 10,000 U pack (03335402001 5 x 2,000 U)
- 2000 U pack (03335399001)
Mfr. no.: Roche
Parameter: 25-55 ° C optimum reaction temperature.
pH range: 5.0 – 9.0
Sent in: dry ice
Storage temperature: −20 ° C
General description
Protector RNase Inhibitor inactivates a broad spectrum of RNases, including
- RNase A
- RNase B
- RNase T2
Therefore, Protector RNase Inhibitor can help prevent RNase degradation in any application where RNases can cause problems. For example, you can:
- protect mRNA during cDNA synthesis reactions, RT-PCR (in conventional thermal cyclers and qPCR systems) or in vitro transcription/translation reactions
- protect viral RNA during virus replication in vitro
- inhibit RNases during RNA isolation and purification
- be used in RNase protection assays
- help prepare RNase-free antibodies
Note: Protector RNase Inhibitor does not interfere with the enzymes commonly used to prepare or analyze RNA.
Content
Protector RNase Inhibitor (40 U / μl) in storage buffer: 20 mM HEPES-KOH, 50 mM KCl, 8 mM dithiothreitol, 50% (v / v) glycerol, pH approximately 7.6 (+ 4 ° C) .
Specificity
- The protective RNase inhibitor definitely does not inhibit RNase T1 and RNase 1.
- Protective RNase inhibitor (20 U / 20 μl reaction) inhibits RNase A up to 1 ng, RNase B up to 160 pg, and RNase T2 up to 0.03 U / μl reaction volume.
- RNase H is not inhibited by Protector RNase Inhibitor.
- Heat inactivation:> 65 ° C
Application
Protective RNase Inhibitor can be used to:
- Protect mRNA in cDNA synthesis reactions, RT-PCR (in conventional thermal cyclers and qPCR systems), for example, with LightCycler® instruments, in vitro transcription/translation system, RNase protection assay, in vitro RNA synthesis.
- Protect viral RNA during virus replication in vitro.
- Inhibits RNases during RNA isolation and purification.
- Helps prepare RNase-free antibodies
- Synthesize RNA Probes for In Situ Hybridization
It is useful in any application where RNases could be a potential problem.
Features and Benefits
- Rely on a thermostable RNase inhibitor: The protective RNase inhibitor remains stable even when using thermostable reverse transcriptases like Transcriptor Reverse Transcriptase.
- Benefit from a wide range of reaction conditions: Protector RNase Inhibitor is active at a pH of 5.0 to 9.0 and at temperatures between + 25 ° C and + 55 ° C (the partial activity can still be measured at + 60 ° C).
- Eliminate interference in different RNA analysis applications – Maintain performance even when adding 16 times more than the standard concentration.
- Insist on a highly purified preparation: the function of each lot is tested using techniques such as quantitative RT-PCR to ensure the absence of endonucleases, ribonucleases or cleavage activity, according to current quality control procedures.
Quality
The function of each lot of Protector RNase Inhibitor is tested with the Titan One Tube RT-PCR Kit and the LightCycler DPD Kit. The protective RNase inhibitor is also tested for contaminating activities as described below.
- Assay buffer: 50 mM Tris-HCl, 10 mM MgCl2, 0.1 mM EDTA, 7 mM β-mercaptoethanol; pH 7.5 (+ 37 ° C).
- Absence of endonucleases: 1 µg of EcoR I / Hind III * fragments of λDNA are incubated with Protector RNase Inhibitor in 50 µl of test buffer at + 37 ° C for 1 hour. The amount of inhibitor that does not cause alteration in the banding pattern is indicated under “Endo”.
- Absence of shear activity: 1 µg of supercoiled pBR322 DNA is incubated with Protector RNase Inhibitor in 50 µl of test buffer at + 37 ° C for 1 hour. The amount of inhibitor that does not cause relaxation of the supercoiled DNA is indicated in “Nick. Act.”.
- Ribonuclease absent (1): 5 µg of MS2 RNA is incubated with Protector RNase Inhibitor for 1 hour at + 37 ° C in a final volume of 50 µl. The amount of inhibitor that does not cause degradation of MS2 RNA is indicated under “RNase 1”.
- Ribonuclease absent (2): 5 µg of MS2 RNA is incubated with Protector RNase Inhibitor for 1 hour at + 37 ° C, then 10 minutes at + 65 ° C in a final volume of 50 µl. The amount of inhibitor that does not cause degradation of MS2 RNA is indicated under “RNase 2”.
Definition of unity
One unit of Protector RNase Inhibitor is defined as the amount of protein necessary to inhibit 50% of the 5 ng activity of RNase A.
Unit Assay: Activity is measured according to Blackburn as the ability to inhibit hydrolysis of cyclic cytidine-2 ‘: 3’-monophosphoric acid. Under the assay conditions, 200 U of Protector RNase Inhibitor inhibits 50% of the activity of 1 μg of RNase A.
Volume activity: 40 U / μl
Preparation note
Work concentration:
- 5 to 10 U in one-step RT-PCR
- 25 to 50 U in two-step RT-PCR
- 20 U for in vitro transcription
You can use higher concentrations of Protector RNase Inhibitor in RT-PCR if you suspect that RNase contamination makes certain samples difficult to amplify. The inhibitor does not interfere with the reaction. (In a test system, even a concentration 16 times higher inhibitor did not interfere with RT-PCR.)
