Nayan Guo 英国伦敦大学 伦敦国王学院 中国 | LinkedIn
来自 : 蚂蚁淘
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WeusethePromegaRibomaxLargeScaleRNAProductionSystemT7(Cat.No.:P1300)toproducedsRNAsandtheJackDixonprotocol(downloadedversion,15.1.2003)topreparedsRNAs.
I.PreparationofTemplateDNA
- Designtwooligosforyourgeneofinterest.Eachshouldincorporatea5"T7RNApolymerasebindingsite,resultinginaPCRproductofapproximately700bp.
- T7RNApolymerasebindingsite:TTAATACGACTCACTATAGGGAGG
- PurifythePCRDNAtemplateItshouldbefreeofRNasesandinhibitorssuchashighsalt,detergentsorEDTA.
- QuantifythePCRProductonanagarosegel.ThedsRNAreactiondetailedbelowrequires5-10µgtotalDNAin40µl.
II.PreparationofthedsRNA
WeusethePromegaRibomaxLargeScaleRNAProductionSystemT7.Wemodifythesuppliedprotocolasfollows:
- Preparethetemplatesasdescribedabove.
- Addthefollowingreagentsfromthekitina1.5mlEppendorftubeatRT.Fora100µlreaction:
- 20µlBuffer5x
- 30µlrNTPs(25µM)
- DNA5µg
- 10µlT7enzymemix
- nucleasefreewatertoafinalvolumeof100µl
- Mixtubegently,spincontentsdownandincubateat37˚Cfor2to6hoursorovernight
- Add2µlofRNasefreeDnase.Incubatefor1hat37¾C.Add200µlofnucleasefreewaterandextracttheRNAwithPhenol:Chloroform
- Ethanolprecipitateyourreactions
- ResUSPendthepelletin100µl-200µlwater.
- Heatyourreactionstubesat70˚Cfor30min.
- Placetubesinabeakerof80˚CwateronthebenchandslowlycooltoroomtemperaturetoannealtheRNA.
- AdjustthefinalconcentrationofyourdsRNAto3µg/µl,usingthefollowingcalculation:OD260nm1=45µg/ml
- Run1-2µgofyourdsRNAona1%AgarosegeltochecktheintegrityandsizeofthedsRNA.A100µlreactiontypicallyyieldsupto1mgofdsRNA.
III.RNAiinDrosophilaSchneidercells.
- WashyourDrosophilacellswithserumfreemediumandplatethemintoabigflaskwith2x106cells/ml.
- Add15µgdsRNAtoper106cells(notice:checkforknockdownefficiencyanduseappropriateamountsofdsRNA)
- SwirlflaskswelltomixRNAandcells.
- Incubatefor1-3hoursintheincubator.
- AddthesamevolumeofDrosophilaSchneiderMediumsupplementedwithdoubleamountsofFetalCalfSerum,Penicillin/StreptomycinandGlutaminetobringthemediumbacktonormalgrowthconditions.
- Harvest3-4dayslater,dependingonyourknockdownefficiency.
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