SucroseDensityFractionation

LEVELII

Figure14.2SucrosegrADIentdistributionofRNA

Materials

• 10%and40%(w/v)Sucrose8
• 0.02Msodiumacetate,pH5.1,containing0.1MNaCland1mM
• EDTA
• RNA
• Ultracentrifuge,swingingbucketrotorandtubes
• Centrifugetubefractionatingdevice
• UVspectrophotometer

Procedure

1. RefertoChapterThreefordetailsoffractionation,andforma10-40%linearsucrosegradientinanitrocellulosetube.
   a.Closeallvalvesonthegradientdevice.

   b.Placeexactly15ml.9of40%sucroseintherightchamberand15ml.of10%sucroseintheleftchamberofthegradientdevice.

   c.Opentheflowfromtherightchambertothecentrifugetube.Besurethereisatubeinplace,thattheflowisdirecteddowntheinsideofthetubeandthatthemagneticstirrerisfunctioning.

   d.Immediatelyopenthevalveontheleftchamberandinsurethatsucroseisflowingfromlefttoright,therebydilutingthe40%sucrosewith10%sucrose.

   e.Allowtheflowtocontinueuntilallofthesucroseentersthecentrifugetube.

   • DissolvetheRNAin0.02Msodiumacetatesolutiontoyieldafinalconcentrationof250µg/ml.ThelowpHofthesolutionhelpstoinhibitRNAse,whilethesaltswillkeeptheRNAfromforminglargepolymericaggregates.10

   • Carefullylayer2.0mlofthedissolvedRNAontothetopofasucrosegradient.Thisshouldbedonebyslowlyallowingthesolutiontorundownthesideofthetubeandontothegradient.Becarefulnottodisturbthegradient.

   • Loadtheultracentrifugewiththepreparedtubesandcentrifugefortheequivalentof18,700RPMforaBeckmanSW27rotor,for20hoursat4°C(RefertoAppendixF).

   • Atthecompletionofcentrifugation,removethetubesandfractionatethecontentsinto1.0mlfractions.
     a.Insertthenitrocellulosecentrifugetubeintotheplexiglassholder,butbecarefulNOTtopuncturethebottom.

     b.Haveaseriesof30-35testtubesreadytoaccepttheeffluentfromthetubes.Eachtubeshouldbelabeledandkeptinorder.

     c.Pushdownonthecentrifugetube(Gently!)inordertopuncturethebottomofthetubeandimmediatelybegintocollecttheeffluent.

     d.Countthedropsthatfallfromthedevice,andplace40dropsintoeachtube.

     • UsingaUVspectrophotometer11andmicrocuvettes,readtheAofeachfraction.CalculatetheamountofRNAineachfraction.

     • PlottheconcentrationofRNAineachfractionagainstthefractionnumber.Basedonthedensityofsucroseineachfraction,computethedensityoftheRNAinthatfraction.Basedontherelativesize(greaterdensity)oftheRNA,determinethenatureofthefractions(i.e.rRNA,tRNA).
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