Sucrose Density Gradient Fractionation 实验方法
来自 : 蚂蚁淘
SucroseDensityFractionationLEVELII
Figure14.2SucrosegrADIentdistributionofRNAMaterials
- 10%and40%(w/v)Sucrose8
- 0.02Msodiumacetate,pH5.1,containing0.1MNaCland1mM
- EDTA
- RNA
- Ultracentrifuge,swingingbucketrotorandtubes
- Centrifugetubefractionatingdevice
- UVspectrophotometer
Procedure
- 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,readtheA
ofeachfraction.CalculatetheamountofRNAineachfraction. - PlottheconcentrationofRNAineachfractionagainstthefractionnumber.Basedonthedensityofsucroseineachfraction,computethedensityoftheRNAinthatfraction.Basedontherelativesize(greaterdensity)oftheRNA,determinethenatureofthefractions(i.e.rRNA,tRNA).
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