Description:
ShortCut?RNaseIII,usedwithitsmanganese-containingreactionbuffer,convertslongdouble-strandedRNAintoaheterogeneousmixofshort(18–25bp)interferingRNAs(siRNA)suitableforRNAinterferenceinmammaliancells(1–3).1.5units(1?μl)ofShortCutRNaseIIIissufficienttoconvert1μgofdsRNAintosiRNAsuitableforRNAinterferenceinmammaliancells.
 ShortCutRNaseIIIdigestionofdsRNA:(A)VaryingamountsofShortCutRNaseIIIwereincubatedwith2μgofa500bpdsRNAfor20minutesat37°Cina50μlreaction.Digestswereanalyzedby20%TBEpolyacrylamideelectrophoresis.Markerlanecontainsamixtureof21bpsiRNAMarkerand100bpDNALadder(NEB#N3231).(B)dsRNAfragments(1kband175bp)weredigestedwithShortCutRNaseIII.Digestswereanalyzedby20%TBEpolyacrylamidegelelectrophoresis.
ShortCutRNaseIIIdigestionofdsRNA:(A)VaryingamountsofShortCutRNaseIIIwereincubatedwith2μgofa500bpdsRNAfor20minutesat37°Cina50μlreaction.Digestswereanalyzedby20%TBEpolyacrylamideelectrophoresis.Markerlanecontainsamixtureof21bpsiRNAMarkerand100bpDNALadder(NEB#N3231).(B)dsRNAfragments(1kband175bp)weredigestedwithShortCutRNaseIII.Digestswereanalyzedby20%TBEpolyacrylamidegelelectrophoresis. GFPSilencinginCOS-7Cells:COS-7cellsco-transfectedina24wellplatewithaplasmidexpressingGFPintheabsence(control)orthepresenceof30ng(4nM)ofGFPsiRNApreparedusingtheShortCutRNAiKit.Cellswerephotographed48hourspost-transfection.
GFPSilencinginCOS-7Cells:COS-7cellsco-transfectedina24wellplatewithaplasmidexpressingGFPintheabsence(control)orthepresenceof30ng(4nM)ofGFPsiRNApreparedusingtheShortCutRNAiKit.Cellswerephotographed48hourspost-transfection.
Highlights
- Isolatedfromarecombinantsource
- Genesilencing
- Targetvalidation
- Suppliedwith10XReactionBuffer
ProductSource
AnE.colistraincontainingageneticfusionoftheE.coliRNaseIIIgene(rnc)andthegenecodingformaltosebindingprotein(MBP).ReagentsSupplied
Thefollowingreagentsaresuppliedwiththisproduct:
|  | Storeat(°C) | Concentration | 
| 10XEDTA |  | 500mM | 
| MnCl2 |  | 10X | 
| GlycogenRNase-Free |  | 10mg/ml | 
| ShortcutReactionBuffer |  | 10X |