u2os drgfp hr.docx

u2os drgfp hr.docx

《u2os drgfp hr.docx》由会员分享，可在线阅读，更多相关《u2os drgfp hr.docx（3页珍藏版）》请在冰豆网上搜索。

U2OSDRGFPHR

Experiment:

measuresoveralldefectsinDSB-inducedHR

Cells:

U2OSDRGFP（DRGFP-purorandomlytargetedandconfirmedforsingleintegrationbysouthern）

Thawcells（PrepareinTChood）:

1.5mLU2OSmediaina15mLscrew-captube（falcon）

2.Add6mLofmediato10cmTCplate.

3.Removetubefromliquidnitrogenfreezer.Thegoalistothawquicklyandtreatthecellscarefullywhenthey'vefirstthawedout.Swirlthetubein37°Cwaterbathuntilmelted.

4.Underhood,changethepipetterto“slow”speed.Remove2mLofthemediafromthefalcontubeandslowlyaddthemediatothecryotube,mixingthecellswithmedia.Slowlyaddthemixedmediawithcellsbacktothe15mLfalcontube,slowlypipettingupanddowntomix.

5.Spincellsinthetable-topcentrifuge3minutesat1000rpm.Iftherearefewcells,spininswingingbuckettopelletcellsatthebottomofthetube.

6.Aspirateoffsupernatant.Add2mLoffreshmediaandslowlypipetteupanddowntomix.Thiswillbreakupanyclumps.Ideallyyouwantsinglecells.

7.Addcellsandmediato10cmplate.Mixbyswirlingtoensureevencoverageonthebottom.

8.Lookatcellsat4Xor10Xscopetomakesurecellshavebrokenup.

9.Moveplatetoincubator.

10.Thenextday:

checkcells-ifconfluent,split1:

5to2x15cmplates

11.U2OScellsgenerallyneedtobesplitevery2-3daysat1:

3to1:

5.

Day0:

splitcellsfromaconfluent15cmplate1:

3to2x15cm:

1.Aspirateoffmedia.

2.washwith10mLPBS;aspirateoffPBS

3.add3mLtrypsin（warmedto37°C）toeachplate.

4.incubateat37°Cfor3-5minutesoruntilcellsbecomedetached）

5.whilecellsareincubating,preparenewplatestosplittobyadding20mLpre-warmedmedia

6.add6mLmediatoeachplatewithtrypsinizedcells

7.resuspendcellstomakesingle-cellsuspension

8.add3mLofcellsuspensionto2new15cmplateswith20mLofprewarmedmedia.

9.placeinincubators

Day1:

DNATransfectionsPreparecells:

U20SDRGFPHRassay

J.LaRocque,adaptedfromJasinlab

06/01/2011

1.trypsinizecellsasonDay0

2.poolcellsinto50mLfalcontube

3.dilute100uLcellsuspensioninto500uLPBS

4.usedilutedcellsuspensiontocountcellsusinghemocytometer

5.resuspendcellsinOPTI-MEMorPBSto7.7x106cells/mL

PrepareDNA:

add25pigofplasmidintoeachcuvette:

A:

CAGGS（noDSBcontrol）

B:

CBAS（inducesDSB）

C:

NZE-GFP（measurestransfectionefficiency）

Electroporatecells:

1.add0.650mLofresuspendedcellsintoeachcuvette（5x106cellseachtransfection）

2.setBio-RadGenePulsarIIelectroporator:

250V;950|iF

3.Pulse,add0.5mLmediatocuvette,thentransferto10cmplatewith8mLmedia

4.Putinincubatorfor48hours

Day3:

（48hrsposttf）:

FACSanalyses

Preparecells:

1.trypsinizecellsaspreviously,usingonly1mLtrypsinfor10cmplates

2.add2mLmediaandresususpendcellsintosingle-cellsuspension

3.transfer1mLofcellsuspensionintoFACstubes（Falcon#352008）.FACsfrommedia;cannotfixcellsduetoGFPleakagefromcells,somustFACswithinanhour.

FACScells:

atapprox100-400cells/second.

FACScansettings:

Detectorvoltageampgainmode

FSC E-1 4.82 LOG

SSC 228 1.0 LOG

FL1 461 LIN

FL2 549 LIN

FL3NA

FL210%FL1

Reeagentsandmaterials:

U2OSmedia:

500mLDMEM-HG

50mLFetalBovineSerum（GeminiBio-Products;cat#100-106althoughanyFBSshouldbefine）

5.5mLPen-StrepSolution100X（GeminiBio-Products）

U20SDRGFPHRassay

J.LaRocque,adaptedfromJasinlab06/01/20111XPBS（noMgorCa）

Trypsin0.2%;ImMEDTAinPBSw/oMgorCaw/ophenolred

Electroporationcuvettes（Bio-Rad0.4cm;cat#1652088）

15cmplates（Nunc#12-565-100）

10cmplates（BDFalcon#08-772E）

15mLfalcontube

50mLfalcontube

5mLtubesforFACS（falcon#352008）

hemocytometer

50mLfalcontube

OPTI-MEM（invitrogen）-CanalsousePBSisyoudon'thaveOPTI-MEMinBioRadGenePulserII