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Minipreps

Stuffyouneed:

    TENSBuffer
      10mMTris-HClpH8.01mMEDTApH8.00.1NNaOH0.5%SDS
    TEpH8.0
      10mMTris-HClpH8.01mMEDTApH8.0
    95%EtOHprecooledto-20degC
    70%EtOHatroomtemperature
    3MNaAcetatepH5.2
  1. Spin1.5mlofovernightculturefor10sinamicrocentrifugetopelletcells.
  2. Decantsupernatant,leaving50-100µlofmediawithpellet.VortexthoroughlytoresUSPendcells.
  3. Add300µlTENS,vortexonhigh2-5s.
  4. Add150µl3MNaAcetatepH5.2,vortexonhigh2-5s.
  5. Spin4mintopelletcelldebrisandchromosomalDNA.
  6. Transfersupernatant(~450µl)toafreshtube(youcanusuallydothisbysimplypouringthesupernatant)andmixwellwith0.9ml95%EtOHwhichhasbeenprecooledto-20degC.
  7. NOTE:IfthestrainyouareworkingwithisnotendA-(e.g.MC1066,JF1754orTG1/lamBDa),itisrecommendedtodoa1:1phenol:chloroformextractionbeforeaddingtheethanol.
  8. Spin2minatroomtemperaturetopelletplasmidDNA.
  9. Discardsupernatant,andwashtwicewith70%EtOH(roomtemperature).Washingsimplyinvolvesgentlyaddingsome70%EtOH(usingasquirtbottle,forexample),andthenpouringitoff.
  10. Resuspendpelletin200µlTEpH8.0
  11. Use10µlperdigestandbesuretouseRNaseduringdigestion.


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