COLONYHYBRIDIZATION

1)CUT4PIECESOF3MMWHATMANPAPERANDPLACEEACHONEINASEPARATECONTAINER(ACAFETERIATRAYWILLPROBABLYWORKPERFECTLY).

2)SATURATEEACHOFTHE4PIECESWITHONEOFTHEFOLLOWINGSOLUTIONS:A)10%SDSB)DenaturingSolution(.5NNaOH,1.5MNaCl)C)Neutralizingsolution(1.5MNaCl,.5MTrispH7.4)D)2XSSC

**DONOTALLOWTHEFILTERSTOBECOMETOOWETBECAUSETHECOLONIESWILLSWELLANDDIFFUSEDURINGLYSIS.

3)PLACETHENITROCELLULOSEFILTERSCOLONYSIDEUPONTHESDSSATURATEDPAPER.ALLOWTOINCUBATE3MINUTES.

**YOUMAYWANTTOTRANSFERTHEFILTERSTODRYWHATMANPAPERBETWEENTREATMENTS.

4)TRANSFERTODENATURATIONSATURATEDPAPERFOR5MINUTES.

5)TRANSFERTONEUTRALIZATIONSATURATEDPAPERFOR5MINUTES.

6)TRANSFERTO2XSSCSATURATEDPAPERFOR5MINUTES.

7)ALLOWTODRYATRT30MINUTES.CROSSLINK.

PREWASH

1)FLOATBAKEDFILTERSON2XSSC,ONCECOMPLETELYWETSUBMERGE5MINUTES.

2)TRANSFERTOPREWASHSOLUTION5XSSC.5%SDS1mMEDTAPH8

3)GENTLYSCRAPEBACTERIALDEBRISFROMSURFACEOFFILTERSUSINGKIMWIPESSOAKEDINPREWASHSOLUTION.INCUBATEAT50DEGREESFOR30MINUTES.

4)TRANSFERTOPREHYBSOLUTION.