.

WeusethePromegaRibomaxLargeScaleRNAProductionSystemT7(Cat.No.:P1300)toproducedsRNAsandtheJackDixonprotocol(downloadedversion,15.1.2003)topreparedsRNAs.

I.PreparationofTemplateDNA

1. Designtwooligosforyourgeneofinterest.Eachshouldincorporatea5"T7RNApolymerasebindingsite,resultinginaPCRproductofapproximately700bp.
2. T7RNApolymerasebindingsite:TTAATACGACTCACTATAGGGAGG
3. PurifythePCRDNAtemplateItshouldbefreeofRNasesandinhibitorssuchashighsalt,detergentsorEDTA.
4. QuantifythePCRProductonanagarosegel.ThedsRNAreactiondetailedbelowrequires5-10µgtotalDNAin40µl.

II.PreparationofthedsRNA

WeusethePromegaRibomaxLargeScaleRNAProductionSystemT7.Wemodifythesuppliedprotocolasfollows:

1. Preparethetemplatesasdescribedabove.
2. Addthefollowingreagentsfromthekitina1.5mlEppendorftubeatRT.Fora100µlreaction:
   • 20µlBuffer5x
   • 30µlrNTPs(25µM)
   • DNA5µg
   • 10µlT7enzymemix
   • nucleasefreewatertoafinalvolumeof100µl
3. Mixtubegently,spincontentsdownandincubateat37˚Cfor2to6hoursorovernight
4. Add2µlofRNasefreeDnase.Incubatefor1hat37¾C.Add200µlofnucleasefreewaterandextracttheRNAwithPhenol:Chloroform
5. Ethanolprecipitateyourreactions
6. ResUSPendthepelletin100µl-200µlwater.
7. Heatyourreactionstubesat70˚Cfor30min.
8. Placetubesinabeakerof80˚CwateronthebenchandslowlycooltoroomtemperaturetoannealtheRNA.
9. AdjustthefinalconcentrationofyourdsRNAto3µg/µl,usingthefollowingcalculation:OD260nm1=45µg/ml
10. Run1-2µgofyourdsRNAona1%AgarosegeltochecktheintegrityandsizeofthedsRNA.A100µlreactiontypicallyyieldsupto1mgofdsRNA.

III.RNAiinDrosophilaSchneidercells.

1. WashyourDrosophilacellswithserumfreemediumandplatethemintoabigflaskwith2x10⁶cells/ml.
2. Add15µgdsRNAtoper10⁶cells(notice:checkforknockdownefficiencyanduseappropriateamountsofdsRNA)
3. SwirlflaskswelltomixRNAandcells.
4. Incubatefor1-3hoursintheincubator.
5. AddthesamevolumeofDrosophilaSchneiderMediumsupplementedwithdoubleamountsofFetalCalfSerum,Penicillin/StreptomycinandGlutaminetobringthemediumbacktonormalgrowthconditions.
6. Harvest3-4dayslater,dependingonyourknockdownefficiency.

================  蚂蚁淘在线  ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。