Product Class: Nuclease

ShortCut® RNase III

recombinant unique buffer incubation temp heat inactivation no
Catalog #SizeConcentration
M0245S200 units2,000 units/ml
M0245L1,000 units2,000 units/ml

Description

Highlights

  • Isolated from a recombinant source
  • Gene silencing
  • Target validation
  • Supplied with 10X Reaction Buffer

ShortCut® RNase III, used with its manganese-containing reaction buffer, converts long double-stranded RNA into a heterogeneous mix of short (18–25 bp) interfering RNAs (siRNA) suitable for RNA interference in mammalian cells (1–3). 1.5 units (1 µl) of ShortCut RNase III is sufficient to convert 1 µg of dsRNA into siRNA suitable for RNA interference in mammalian cells.

ShortCut RNase III digestion of dsRNA: (A) Varying amounts of ShortCut RNase III were incubated with 2 µg of a 500 bp dsRNA for 20 minutes at 37°C in a 50 µl reaction. Digests were analyzed by 20% TBE polyacrylamide electrophoresis. Marker lane contains a mixture of 21 bp siRNA Marker and 100 bp DNA Ladder (NEB #N3231). (B) dsRNA fragments (1 kb and 175 bp) were digested with ShortCut RNase III. Digests were analyzed by 20% TBE polyacrylamide gel electrophoresis.
GFP Silencing in COS-7 Cells: COS-7 cells co-transfected in a 24 well plate with a plasmid expressing GFP in the absence (control) or the presence of 30 ng (4 nM) of GFP siRNA prepared using the ShortCut RNAi Kit. Cells were photographed 48 hours post-transfection.

Product Source

An E. coli strain containing a genetic fusion of the E. coli RNase III gene (rnc) and the gene coding for maltose binding protein (MBP).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
10X EDTA500 mM
MnCl210X
Glycogen RNase-Free10 mg/ml
Shortcut Reaction Buffer10X

Advantages and Features

Applications

  • Gene silencing
  • Target validation

Properties and Usage

Unit Definition

One unit is the amount of enzyme required to digest 1 µg of dsRNA to siRNA in 20 minutes at 37°C in a total reaction volume of 50 µl.

Reaction Conditions

1X ShortCut Reaction Buffer
Incubate at 37°C

Usage Concentration

1X

Storage Temperature

-20°C

Storage Conditions

500 mM NaCl
10 mM Tris-HCl
0.5 mM EDTA
1 mM DTT
pH 8.0 @ 25°C

Heat Inactivation

No

Unit Assay Conditions

One unit is the amount of enzyme required to digest 1 μg of dsRNA to siRNA in 20 minutes at 37°C in a total reaction volume of 50 μl.

Heat Inactivated

No

Supporting Documents

Material Safety Datasheets

The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

References

  1. Morlighem, J.E. et al. (2007). Biotechniques. 42, 599-606.
  2. Yang, D. et al. (2002). Proc. Natl. Acad. Sci. USA. 99, 9942-9947.
  3. Calegari, F. et al. (202). Proc. Natl. Acad. Sci. USA. 99, 14236-14240.
  4. Donze, O. and Picard, D. (2002). Nucleic Acids Res. 30, e46.
  1. What products does NEB offer for functional genomics and reverse genetics?
  1. ShortCut RNase III Digestion Protocol (M0245)
  2. Purification of siRNA by Ethanol Precipitation Protocol (M0245)
  3. Transfection Guidelines for ShortCut® RNase III (M0245)