Northern Blot: Glyoxal/DMSO Northern Blotting 资讯 生物在线
来自 : 蚂蚁淘
NorthernBlot:Glyoxal/DMSO
AdaptedfromMolecularCloning(Maniatis),CurrentProtocolsinMolecularBIOLOGy,andJ.YostLab(UMN)
Objective:
Glyoxal/DMSOgelsaregreatfordeterminingsizesofRNAespeciallyinconjunctionwithnorthernblots.Procedures:
Start=>RNA,eitherisolatedtotalRNA,poly(A+)RNA,orinvitrotranscribedRNA.- Beginbypreparingthesampleforheatdenaturation.Combineallofthereagentsfromthefollowingtableexcept10XloADIngbufferinmicrocentrifugetubes.Ifmanysamplesarebeingprepared,itmaybemoreefficienttomakeamastermixofNaPO4,DMSO,andglyoxal.Tomakethemastermix,multiplytheamountofeachcomponentrequiredpersamplebythenumberofsamples+10%(don"tforgettheladder).Thechoiceofsamplevolumedependsonwell-sizeandRNAconcentration.
| PreperationofRNAsample |
|---|
| Reagent | For15uL | For20uL | For25uL | For30uL |
|---|
| RNA(upto10ug) | 3uL | 4uL | 5uL | 6uL |
| 0.2MNaPO4pH7.0 | 0.75uL | 1uL | 1.25uL | 1.5uL |
| DMSO | 7.5uL | 10uL | 12.5uL | 15uL |
| 6M(40%)Glyoxal* | 3uL | 3.75uL | 2.25uL | 4.5uL |
| 10XLoadingBuffer** | 1.5uL | 2 | 2.5uL | 3uL |
| *Freshlydeionized(seetext). |
| **Addafterheatdenaturation(seetext). |
- Mixthesamplesbytappingtheendofthemicrofugetubewithyourfinger.Thenbrieflypulsethesamplesdowninamicrocentrifuge.Incubateat50degreesforonehour.
- Whilethesamplesareincubating,preparethegel:dissolve0.8gagarosein100mLof10mMNaPO4pH7.0(5mLof0.2MNaP04pH7.0+95mL"DEPC"H2O).MakesuregelboxandcombareRNase-freebytreatmentwith3%H2O2orcommericialRNaseremovalproduct(e.g.RNaseZapfromAmbion).
- Aftersamplesaredoneincubating,placeoniceandaddloadingbuffer.MixsampleswithPipettetip,andifnecessarypulsedowninmicrocentrifuge.
- PlacesolidifiedgelinRNase-freegelbox,andcoverwith10mMNaPO4pH7.0runningbuffer.
- Loadsamplesandrunat4V/cmuntilbromphenolbluehasmigratedapproximately8cms(approximately3hours).Recirculationoftherunningbuffer(fromtheannodetothecathode)isrequiredtopreventaH+gradientandsubsequentdissociationofglyoxal.
- Removegel.
- Tovisualize,placeallorportionofgeltobevisualizedinRNase-freedishandstainwith0.5ug/mLEtBrin0.5Mammoniumacetatefor~20-40minutes.Thendestainwith0.5Mammoniumacetateforanadditional~20-40minutes.Alternativelytheladdercanbestainedaftergeltransferusingmethyleneblue.
Reagents/Solutions
Preperationof6M(40%)Glyoxal
- DeionizeglyoxalwithBio-RadAG501-X8(orX8D)resinuntilthepHis5.0-5.5.Dothisimmediatelybeforeuse,orafterdionization,storeinsingle-usealiquots(nofreeze-thaw-freeze)insmalltightlycappedtubes.
10XLoadingBuffer
- 50%glycerol
- 10mMNaPO4pH7.0
- 0.25%bromophenolblue
- 0.25%xylenecyanolFF
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