DNAgelpurification(UltraCleanTM15Kit):

1. CutthegelbandunderUVlight(2wellsfor50uldigestion).
2. Add1mlofULTRASALTandmeltthegelat5565Cfor10min(mixoccasionallyto
makesurethegelmeltcompletely).
3. Add6ulofULTRABIND(resuspendthoroughlybyvortexing),andmixwell.Incubatefor
10minatRT(mixseveraltimes).
4. CF15secondsandremovesupernatant.
5. Resuspendthepelletin1mlofULTRAWASH.
6. CF15secondsandremovesupernatant.WashwithULTRAWASHonemoretime.
7. Vacuumdrythepelletfor3min.
8. Resuspendthepelletin30ulofH2OorTE(disturbthepelletwiththepipettetipto
resuspend).
9. CF1minandremovethesupernatant(DNAinthesupernatant).

Ligation:

5XBuffer
2ul
Insert(gelpurified)
3ul
Vector (gelpurified)
1ul
10mMATP
0.5ul
T4Ligase
1ul
H2O
2.5ul
MixwellandkeepatRT1ho/n.

Transformationandplating:
1.
2.
3.
4.
5.
6.
7.

Thawcompetentcellsonice
Mix5uloftheligationreactionand50ulofcellsin1.5microfugetube.
Incubateonicefor2030min.
Heatshockthecellsat42Cfor3045seconds.
Placethecellsonice.
Add500mlofLBmediumtothecellandshakethetubeat37Cfor45minto1h.
Spread150ulcultureontoLBplatecontainingproperantibiotics.Incubateo/n
at37C.
Or Pelletcellsat5000rpmfor1minandquicklypourofftheLB(about100150ul
leftoverLB+pelletstillinthetube).Resuspendthepelletandplateentire
contentsontLB+properantibioticsplates.

TOPOTACloning(kit):
PCRreaction:
Buffer(withMg)
5ul
DNA(template)
0.5ulofMiniprepDNA(100500ug)
dNTPs(10mM)
1ul
3primer(50pmoles/ul)
0.5ul
5primer(50pmoles/ul)
0.5ul
Enzyme(DyNAzyme)
0.5ul
H2O
42ul
Add23dropsofmineraloil,usehotstart.Heatupto95Cfor1min,thengoto:
94C
30
55C
30(Tmofprimersshouldbe>60C,ifnotlowerannealingT)
72C
1(forlongfragment,increase1/kb)
n=35
Linkto72C10,thenkeepat4C.Take25ulrungel.
TOPOligation(kit):
PCRproduct
2ul
SaltSolution
1ul
TOPOvector
0.5ul
H2O
1.5ul
Mixandincubatefor15minatRT,thenplacethetubeonice.
Transformationandplating(kit):
1.
ThawOneShotE.colionice.
2.
Add2uloftheligationreactionto50ulcompetentcellandgentlymix.
3.
Incubateonicefor15min.
4.
Heatshockthecellsat42Cfor30seconds.
5.
Placethecellsonice.
6.
Add250ulofRTSOCmediumtothevial,andshakethetubeat37Cfor1h.
7.
Spread100ulcultureontoLBplatecontainingantibioticsand/orXGal(spread50ulof
20mg/mlontheplateandletitdrybeforespreadthecells).
8.
Incubateo/nat37C.
9.
Pickwhiteorlightbluecolonies(ifuseXGal)forMiniprep.

MiniPrep(alkalinelysis):
1.
Pour1.5mlofano/ncultureintoamicrotube,CFfor1minanddiscardthesupernatant.
2. Resuspendthecellsin100ulofSolution1byvortexing.
3. Add200ulofSolution2,mixbyinvertingthetubestwiceandincubatefor2minatroomT.
4.
Add150ulofcoldSolution3,mixbyinvertingtwiceandincubatefor5minonice.
5.
CFfor5minandcarefullypipettethesupernatant(380ul)inanewtube.
6.
Add1ml100%ethanol,inverttwiceandCFfor2min,discardtheliquid,washthepellet
with70%ethanolandvacuumdrythepelletfor5min.
7.
Resuspendthefinalproductin50ulTEwithRNaseA(20ug/ml).
8.
Take2ultodoenzymedigestion.
SolutionI(4C):50mMglucosein50mMTrisHClpH8.0,10mMEDTA,orinregularTE
(10mMTrisHCl,1mMEDTA,pH7.58.0).
SolutionII(RT):0.2NNaOH,1%SDS.
SolutionIII (4C):3MpotassiumacetateadjustedtopH5.5withglacialaceticacid(100ml
SolutionIII:60ml5MKAc,11.5mlglacialHAc,28.5mlH2O).
Note:Afterstep5,candirectlyload15ul/well(noneedforloadingbuffer,norladdereither)
ontoagelandrunfor25minat150Vtocheckifwegotinsert(thebiggerplasmid).Thisis
goodforlargenumberselection.

Enzymedigestion(forMiniorMidipreps):
PlasmidDNA
10Xbuffer
Enzyme
H2O

2ul
1ul(makesuretousetherightbufferfortheenzyme)
0.2ul
6.8ul(tomakeuptotalvolumeof10ul)

Incubateat37C(someenzymeneedsspecialT)for30min.Loadtotaldigestiononthe
geltocheckifyoucangettherightgelband(goodluck).

MiniPrep(lysozymeboilinglysis):
1.
2.
3.
4.
5.
6.
7.
8.
9.

Pour1.5mlofano/ncultureintoamicrotube,CFfor1minanddiscardthesupernatant
completely.
Resuspendthecellsin200ulofSTETlysozyme(mix180ulofSTETand20ulof
lysozymebeforeuse)byvortexingandkeepatRTfor5min.
Placethetubesinboilingwaterfor1min.
CFfor10minatmaxspeed.
Removethecelldebriswithatoothpick.
Take8ulofsupernatanttorunthegeltocheckitcontainsinsertornot.
Forthoseplasmidswithinsert:
AddH2Otomakeupto200ul,thenadd200ulofisopropanolandmixwellbyinverting
tubesseveraltimes.
CFfor10minatmaxspeed,washthepelletwith70%EtOH,drythepelletand
resuspendin50ulTE+RNase.
Take2ultodoenzymedigestion.

Solutions(storeat4Cseparately):
STET:(10%sucrose,50mMTrisHCl,pH8.0,50mMEDTA,0.2%TritonX100).
Lysozyme:20mg/mlinSTET.

MidiPrep(BioRADkit):
1.

Inoculateplasmidcontainingbacteriainto40mlofLBcontainingproperantibioticsina
flask.Cultureo/nat37Cinarotaryshaker.
2. CFfor5mininatubewithcap,anddiscardthesupernatant.
3. Add5mlofCellResuspensionSolutionandvortextoresuspendthepellet.
4. Add5mlofCellLysisSolutionandinverttwice(donotvortex).
5. Add5mlofNeutralizationSolution,inverttwiceandsitonicefor5min.
6. CFfor10minat10,000rpmandcarefullypourthesupernatanttoanewtube.
7. Add800ulofresuspendedQuantumPrepMatrixandmixwell.KeepthetubeatRTfor
10min(mixfromtimetotime).
8. CFfor2minat8000rpmandpouroffthesupernatant.Add10mlofWashingBufferand
resuspendthematrix.
9. CFfor2minat8000rpmandpouroffthesupernatant.Add10mlofWashingBufferand
resuspendthematrix.CFfor2minat8000rpmandpouroffthesupernatant.
10. Add500ulofWashingbufferandresuspendthematrix.
11. Transferthematrixintoaspincolumnwiththetipsnappedoffsittinginsidea2mlcollection
tube.PunctureaholeinthecolumncapandCFfor1minat10,000rpm.
12. Discardthebufferfromthetubeandputthecolumnbacktothetube.Add500ofWashing
bufferandCFfor1minat10,000rpm.
13. Discardthebufferfromthetube,putthecolumnbacktothetubeandCFfor2minat
maximumspeed.
14. Transferthecolumntoacleantube,add400ulofH20orTEtothematrix,sitfor5min,and
CFfor2minatmaximumspeed.
15. Take2ultodoenzymedigestionandrunthegeltochecktheconcentration.
Note:Onceinawhile,plasmidcanbelostduringo/nculture.Tomakesureyouhavethe
plasmid,youcantake1mlofo/nculturetodoquickMiniprepandrunaquickgel(without
enzymedigestion)tochecktheconcentration.
Afterstep14,youshouldaddanother200ulofH20orTEtothematrixandsavethecolumn.If
yourfinalconcentrationtoohigh,youcanspindownthisandcombinebothelutions.

AgroInfiltration:
1.
2.
3.
4.
5.
6.
7.

GrowA.tonplatecontainingproperantibioticsfor2daysat28C.
Scrapethecellsinto4mlof10mMMESwith10mMMgCl2.
Vertextoresuspendcells,andtake1mltocheckOD600.
Adjusttheleftover3mlsto0.6(OD600)withaboveMES.
Add0.1Macetosyringone(1.5ul/ml),mixandsitinRTfor3h.
Useasyringetoinfiltratethecellsuspensionfromtheundersideoftheleaf(23leaves/plant).
Marktheedgeoftheinfiltratedareaandkeeptheplantinagrowthroom.
Note:dissolveacetosyringoneinDMSO

MakingcompetentcellsofAgrobacteriumtumefaciens:
1.

Inoculate6mlLBRif100with15ulglycerolstockandovernightshakingat28C.

2.

Inoculate50mlLBRif100in250mlflaskwith5mlofovernightculture.

3.

Growcultureat28Cforabout8htoanOD600of0.6.

4.

Chillcultureonicefor5min.Pelletcellsat4000rpmfor5min.Resuspendin1ml20mMCaCl2.

5.

Make100ulaliquotsandstoreat80C.Use2550ulofcompetentcellsforeachtransformation.

TransformationofAgrobacteriumtumefaciens:
1.

Add5ulpurifiedplasmidto25ulofcompetentcellsina1.5mltubeandmixbygentlytappingthe
tube.Freezecellsinliquidnitrogen.

2.

Thawfrozencellsin37Cwaterbathfor5minandthenadd800ulofLB(noantibiotics).

3.

Incubatecellswithshakingat28Cfor23h.Pelletcellsat5000rpmfor40secandquicklypouroff
theLB(about100150ulleftoverLB+pelletstillinthetube).Resuspendthepelletandplateentire
contentsontoLB+properantibioticsplates.Puttheplatesto28C.

4.

23daysyoushouldseecolonies.

Antibioticsstocksolution
Rifampicin:25mg/mlin100%methanol
Kanamycin:25mg/mlinwater
Tetracycline:5mg/mlin100%ethanol
Spectinomycin:50mg/mlinwater

CompetentCells(NEBM13sequencingmanual)
1.
2.
3.
4.
5.
6.
7.
8.

Inoculate7mlofLBwithonecolonyfromaplateandincubateat37Covernight
withshaking(225rpm).
Add46mlofovernightcultureinto200mlfreshmediaandshakeat37Cuntil
OD600is0.40.6(approximately23h).
Chillonicebriefly.Keepeverythingat4Cfromthispointon.
Pelletcellsat5000rpmfor5minat4C.
Gentlyresuspendcellsin40ml(1/5oftheoriginalculturevolume)oficecold0.1M
MgCl2.Keeponicefor1520min.
Centrifugeat4000rpmfor5minat4C.
Gentlyresuspendcellsin4ml(1/50oftheoriginalculturevolume)oficecold0.1M
CaCl2.
Keeponice12htoestablishcompetency.Thecellsmaybefrozenatthispointby
adding1ml(1/4volume)ofsterile75%glycerolandplacedat80in0.1mlaliquots.
Whenretrievingcells,thawoniceanduseimmediately.

SYNVInoculation:
Grindtissuein0.01MNaPB,pH6.87.0containing1%celiteand0.5%Na2SO3.
Note:weighceliteandNa2SO3,mixwiththebufferbeforeinoculation.

ProteinConcentrationAssay(Bradford:BioRad):
PreparestandardIgG:ourstockis1.43mg/ml
Concentration
0.2
0.4
0.6
0.8
1.0
Ourstock
14
31
42
56
70
H2O
86
69
58
44
30
AddH2Ofirstto13mmglasstube,thenaddIgGstock.

1.2
84
16

mg/ml
ul
ul

Dilutedyesolution:8mldye+32mlH2O(dye:water=1:4)
Prepareyoursample:1to10dilution:10ul+90ulH2Oor1to5dilution:20ul+80ulH2O
Colorreaction:Add5mldiluteddyeintoeachtubeandincubateatroomtemperatureformore
than5min.
Measurement:Visuallycheckyoursampletoseewhatconcentrationrangeitshouldbe.Then
takethreesurroundingstandardconcentrationsplusyoursampletomeasureabsorbanceat595
nm(600nmoktoo).Useyourdiluteddyesolutiontozerothemachine.

PreparationofTNMFHinsectmedium
1.
2.
3.
4.
5.
6.

Get~800mlofwaterandstartstirring.Addonebottleofthepowderedmediumand
keepstirringuntildissolved(donotheat).
Add0.35gofsodiumbicarbonateandadjustthepHto6.2withNaOH.
Add100mlofFBSandmorewatertomakeupto1L.
Filterthrough0.45umfilter(needchangefilterafewtimes,usebigredtubefor
vacuumfrombench).
Filterthrough0.22umfilter(understerilecondition:screwthefiltertoanautoclaved
bottleunderhood,thenfilteringonthebenchwiththevacuum).
Aliquotinto50mlsterileplastictubeunderhoodandstoreat4C.

Typhoon Scanner Usage (pw: plantpath)

1. Wipe the glass window and put your Phosphor screen face down at the corner of A and 1
2. Double click Typhoon Scanner icon
3. Select area (Red corresponding to our Phosphor screen) you want to scan, then click Scan
(save your file into your folder)
4. Open your file with ImageQuant (this program is open all the time)
5. Click Edit Image icon and make adjustment
6. Click File-Save As and save your file as tif file into your flash drive

SDSGELELECTROPHORESIS:(fromRY)
Assemblyofgelapparatus
Castingthegel
1.
Preparetheseparatinggelsolutionbycombiningallreagents(addAPSandTEMEDlast)andpourthe
solutiontothegelplate(thetopofthegelwillbe1cmbelowtheteethofthecomb;ifuse 0.75spacer,
pour3.5mlofgelsolution).
2.

ImmediatelyoverlaythegelsolutionwithwaterorH2Osaturatedisobutanol.

3.

Allowthegeltopolymerizefor45minto1h(youcanseeclear2linesbetweensolutions).

4.

PreparethestackinggelsolutionbycombiningallreagentsexceptAPSandTEMED.

5.

Rinseofftheoverlaysolutioncompletelywithwater.Blotdrytheareaabovetheseparatinggelwith
filterpaper.

6.

AddAPSandTEMEDtothestackinggelsolution,mixandpourthesolutionintothegelplates.Place
thecombinthegelsandwich.

7.

Allowthegeltopolymerizefor3045min.Removethecombandrinsethesamplewellscompletely
withwater.

Sampletreatment
Samplefrominfectedplant:Use1.5mlcentrifugetubelidtocutaleafdisc,add200ulof2Xsample
buffer,grindthediscandplaceinaboilingwaterbathfor10min.Centrifugefor5minandtakethe
supernatanttoloadonthegel.Thissamplecanbestoredat4CorRTforlongtime(donotneedtoboil
beforeloading).
Assemblegelapparatus(besuretofollowmanufacturesinstructionstoavoidleaking)
Afterassemblethegelapparatus,addelectrodebuffer(1X).
Loadingsample
Load1020ulofsampletoeachwell.Load5ulofSDSGELstandard(unstainedforCoomassieand
prestainedforWestern;donotneedtoboil,justthawandload).
Runningthegel
Connecttheelectricwareandrunthegelfor~1hat175V(letdyereachthegelbottom).
Removethegel
Disassemblethegelapparatus,openthegelsandwichandremovethegelcarefully.
Staining
FollowPageBluestainingprotocol.

SeparatingGel(8mlfor2gelswhenuse0.75mmspacer,3.5ml/gel)
15%
12%

10%

7.5%

Water (ml)

1.86

2.68

3.34

3.88

1.5MTrisHCl,pH8.8(ml)
10%SDS
Acrylamide/Bis(ml)
(30%stock,37.5:1)
10%APS
TEMED

2.0
80ul
4.0

2.0
80ul
3.2

2.0
80ul
2.66

2.0
80ul
2.0

80ul
4ul

80ul
4ul

80ul
4ul

80ul
4ul

***ForAPS:Preparefreshandgoodfor1wkatroomC,or1monthat4C***
StackingGel(3mlfor2gels)
Water
0.5MTrisHCl,pH6.8
10%SDS
Acrylamide/Bis(30%stock)

4%
1.8ml
0.75ml
40ul
0.39ml

10%ammoniumpersulfate(APS)
TEMED

RunningBuffer(5Xstock)
Trisbase
Glycine
SDS
Maketo1000mlwithwater.

2XSamplebuffer(total10ml)
Water
0.5MTrisHCl,pH6.8
Glycerol
10%SDS
mercaptoethanol
bromophenolblue

SampleConcentration
Purifiedvirus
~5
Protein
~5

60ul
3ul

15g
72g
5g

3.5ml

2ml(pHrangeshouldbe6.87.5)
2ml

2ml

0.5ml(addbeforeuse)
0.01g

ug/lane
ug/lane

WESTERNBLOT:
1.

Soakalltransfercassetteparts(fiberpad,3MMfilterpaper,nitrocellulosepaper)intransferbuffer.

2.

3.

Assembleinthefollowingorderontotheblackside(current:black>white)ofgelcassette.
a.
Fiberpad
b.
3MMfilterpaper
c.
Gel
d.
Nitrocellulosepaper
e.
3MMfilterpaper
f.
Fiberpad
Keeptheblacksideofcassetteontheblacksideofthetransferapparatus.

4.

Blotat0.25Afor1.5~2h(withcoldtransferbuffer,inacontainerandstirring).

5.

Blocknonspecificsiteswith1520mlof5%drymilkinTBSfor30minonshaker.

6.

AddprimaryAb(1:10004000or2~5ug/ml)intotheblockingbuffer. Incubateovernightonslow
shaker.

7.

Wash3X10mininH2Oonshaker.

8.

IncubatethemembranewithsecondaryAb(makesureuse right Abatproperdilution)inblocking

buffer.Incubate2honslowshaker.

9.

Wash3X10mininH 2Oonshaker. Readytodo Chemiluninescentexposure (step12)or Color

development(step10).

10.

Developinthedyesolution(coverfromlightforabout10min).

11.

Rinsewithwaterandairdrythemembrane.

12.

Drainthemembranebytouchingacorneronapapertowel,thenplaceitonplasticwraponaflat
surface(donotletthemembranedry).

13.

Pipetteathinlayerofsubstratesolution(CDPStarReadytouse,12ml)ontotheblotandincubatefor
5min.

14.

Drainthesubstratesolutionandthenwrapthemembraneinplasticwrap.

15.

PlaceastandardXrayfilmoverthemembraneandexposefor10sec5min.Developthefilm.
Dyesolution:(Foreachblot,add50ulofeachdyeinto12mlofbuffer,pH9.5)
NBT(NitriBlueTetrazolium):50mg/mlin0.7mlDMFand0.3mlH2O
BCIP:25mg/mlin1mlDMF(N,NDimethylFormamide)
10XTBS:0.5MTris,1.5MNaCl,pH7.5
TTBS:1XTBS+0.02%Tween20
Transferbuffer:
25mMTris, 192mMGlycine,
For1L 3.03g
14.41g

20%Methanol
200ml

Dyebuffer: 100mMTris,100mMNaCl,5mMMgCl2,pH9.5
Or 100mMNaHCO3,1mMMgCl2,pH9.5
2Xsamplebuffer:

50200mMTrisHCl,pH6.87.5
SDS:24%
Glycerol:1520%
Bromophenolblue:0.1mg/ml
DTT:200mMorBMe:5%(addfresh)

AGAROSEGELELECTROPHORESIS:
CastingGel(0.81.2%):
Meltagarosethoroughlyin1XTAErunningbuffer(4060mlforsmallgel;100150forbiggel),
coolto5060C,add2ulof10%ethidiumbromide,mixwellandcastgel.
RunningGel:
Take10ulofsample,add12ulofloadingbuffer,mixwellandloadontothegel.Load23ul
ofladder(1KB)inonelaneasstandard.
Runthegelforabout2060minat100V.Takepicture.

LoadingBuffer(6X):
0.25%bromophenolblue
0.25%xylenecyanolFF(somebufferwithoutthisdye)
30%glycerolor40%sucrose
inH2O

Ethidiumbromide:
10%ethidiumbromideinH2O(storedatRTindarkbottle). Gelcanbestainedafterrun
(immersethegelin0.5ug/mlEBfor3045minatRTanddestainisnotneeded).
Caution:EBisapowerfulmutagenandalsomoderatelytoxic.Waregloves!

SequencingwithBigDyeTerminator(V3.1)

1.

In0.2mlPCRtube,preparefollowingmix:
BigDyeReactionPremix(at20C)
BigDye5XBuffer(at4C)
Primer(5pmol/ul)
Template(miniormidi)

2ul
1ul
1ul
1ul

H2O
2.

5ul

Performthermalcycles:
96Cfor4min,then35cyclesof:
96C
50C
60C

20sec
5sec(thisCdependsonprimer)
4min

3.Cleanup:
Spindownthereaction,add10ulH2Otobringthe
volumeto20ul,thendomormalcleanupasfollowsorcolumn
cleanuponnextpage.
a)

Transferto1.5mltube.

b)

Add2ul125mMEDTA,2ul3MNaAc(pH5.4)and50ul
100%EtOH.Mixwellandleavefor15minatRT.

c)

Spin30min(maxrpm,RT).

d)

Washtwicewith300ul70%EtOH.

e)

Drythesampleandsendforsequencing.

Note:IfthetemplateisPCRproduct,weneedtodogel
purificationorgothroughCentricontogetridofanyleftover
primers.

NorthernBlot

PurifyRNA
UseRneasyPlantMiniKit(Qiagen)topurifytotalRNA.Use100mgtissueandthe
finalelutionvolumeis50ul.Ifyouneedtodoquantitativecomparison,youneedtomeasurethe
OD260tomakesureequalloadinglater.Storeat80C.
RunRNAdenaturinggel
1.
Cleangeltray,combandgeltankcarefullytogetridofRNasewithmethodAorB:
A,washwithwater,spraywithScrubbingBubbles,sitforafewminutes,rinsewith
water,spraywithRNaseAway,sitforafewminutes,rinsewithautoclavedwater;B,
soakwithwarm0.1%SDSplus10mMEDTA(pH8.0)formorethan1h,rinsewith
autoclavedwater.PutapieceofSaranwrapunderthegeltraybeforecastingthegel.
2.
Prepare1%agarosegel:MOPS,waterandglasswareneedtobeautoclaved.
Forbigtray
Forminitray
Agarose
2g
0.5g
10XMOPSbuffer
20ml
5ml
Water
146ml
36.5ml
Melttheagaroseandletitcooltoaround60C;thenaddformaldehyde.
Formaldehyde(37%)
34ml
8.5ml
Mixwellandcastthegel.

3.

4.

5.

Runningbufferis1XMOPSbuffer(forminigel,needabout250ml).Loadthe
sampleandrunthegel.
Samplepreparation:
Foreachsample(6ul),preparefollowing:
5X
10X

15X

20X
Formamide
12.5ul
62.5 125
187.5 250
10XMOPSbuffer
2.5ul
12.5 25
37.5 50
Formaldehyde(37%)
4ul
20
40
60
80
EtBr(10mg/ml)
0.1ul
0.5
1
1.5
2
Total
19.1ul/sample

Mixwiththesample,incubateat65Cfor5min,thenchillonice.Add2.5ulloading
buffer(50%glycerolcontaining0.1mg/mlbromophenolblue)andloadontothegel.
TreattheRNAladderthesamewayasforsamples(ladder+EB+LB65C5min).
Runthegelat100Vuntilthedyereachtheend(~1.5h).Afterwards,visualizethe
RNAunderUVlightandtakeapicture.Ifyouloadaladderlane,putthegelovera
UVboxandpuncturetheladderband.Alsotrimacorneroffundertheladderlaneto
marktheorientation.
Placethegelinacontainerwith10XSSPEbufferandgentlyshakefor15min
(optional).Thegelisreadytogotoblot.

TransferRNAtomembrane(refertothediagram)
1.
WearcleanglovestocuttheHybondN+membrane(gelsize)and3MMpaper(4
sheetsofgelsizeandalongoneforbridge)andsoakthemintransferbuffer(10X
SSPE,300ml).
2.
Setupaplatforminaglassdishandusethelong3MMpapertomakeabridgeover
theplatform.UseaPasteurpipettetodriveanyairout(repeatthisforthegel,
membraneand3MMpaper).
3.
Put2sheetsof3MMpaperoverthebridge,thenlaythegel(bottomup)ontop.
CoverthetopoftheglassdishwithSaranwrap,thencutitopenalongtheedgesof
thegelwithablade.
4.
putthemembraneonthegelfollowedbyanother2sheetof3MMpaper.Placea
stackofpapertowels(23inchthick)ontopofthe3MMpaper.
5.
Placeaglassplateandaweight(abottleof800mlwater)ontop.Allowthetransfer
toproceedovernight.
6.
Aftertransfer,turnaroundthemembranewiththegel.Useapenciltomarkthe
ladderbandsonthemembraneandtrimacorneroff.Airdrythemembrane,thenfix
theRNAbyUVcrosslink(autocrosslink).Membranecanbestoredatthispointfor
afewdaysordohybridizationrightaway.

Weight

Glass plate
Paper towel
Membrane
Gel
Saran wrap
Long 3MM paper
Glass dish with
transfer buffer

3MM paper

Glass plate
Support

Probepreparation
UseRediprimeIIRandomPrimeLabellingSystem(Amersham/GEHealthcare)to
preparetheprobe.
1.
2.

UsePCRproductofyourgeneofinterestorcutoutyourgenefromaplasmid.In
eithercase,youneedtogelpurifyyourproduct(finalelutionin30ul)andcheckthe
concentration.
FollowtheProtocoltodothelabeling.

Hybridization
1.
Putyourcrosslinkedmembraneintoahybridizationtube(or50mltubeformini
gel).Add5ml(for50mltube)Ultrasensitivehybridizationbuffer(Ambion,#8670;
warmthebufferupat65Cfirst)andprehybridizefor1hat42C.
2.
Addyourlabeledprobeintothebuffer(besuredonotaddtheprobedirectlytothe
membrane).Hybridizethemembranewiththeprobeovernightat42C.
3.
Washthemembranefor510minwith2XSSPE/0.1%SDSat42C.Washonemore
time.
4.
Washthemembranefor15minwith0.1XSSPE/0.1%SDSat42C.Washonemore
time.
5.
WrapthemembraneinSaranwrap.
6.
ExposethemembranetoPhosphorscreenovernight(orlongerifthesignalisnot
strong).
7.
VisualizetheimageusingaPhosphorImager.
Buffers(needtobeautoclaved)
1.
20XSSPEbuffer:3MNaCL,0.2MNaH2PO4,0.02MEDTA,pH7.4.
2.
10XMOPSbuffer:0.2MMOPS,0.1Msodiumacetate,0.01MEDTA,pH7.0.
3.
Prehybridizationandhybridizationbuffer:Ultrasensitivehybridizationbufferfrom
Ambion(#8670).
4.
20XSSCbuffer(replaceSSPEwithSSCforSouthern):3Msodiumchloride,0.3M
sodiumcitrate,pH7.0

Southernblot(HybridizationanalysisofDNA)
PurifytotalDNAandenzymedigestion
PurifyTotalDNAfromplant.
DigesttheDNAintosmallfragments(use200uldigestionsystem,thenconcentrateto40ul).
Rungel
Loadthesampleon~7mmthick0.7%agarosegelwithEB(loadDNA+alittleloadingbuffer,
rungelfor30minat70V),thensubmergethegeltotallyandrunat~20Vovernight.
Afterrunningthegel(takeafinalpicture),punchtheladderbandsonUVbox,thencutacorner
undertheladderlane.
Depurination(Optional,sincethefragmentslargerthan15Kbarehardtotransfer)
Washthegelin0.2MHClfor5(minigel)to10(largegel)minonashaker,thenrinsewith
water.
Denaturation
Washthegelindenaturationbufferfor30minonashaker,thenrinsewithwater.Transferthe
gelintoneutralizationbufferfor30minonashaker.Nowthegelisreadytogotoblotting.

Blottingandhybridization

SameasinNorthern(useSCCinsteadofSSPE).
Buffers
1.

20XSCCbuffer:3Msodiumchloride,0.3Msodiumcitrate,pH7.0.

2.

Depurinationsolution:0.3MHCl.

3.

Denaturationsolution:1.5MNaCl,0.5MNaOH.

4.

Neutralizationbuffer:1.5MNaCl,0.5MTrizmabase,pH7.5.

Note:Genomic DNA is digested with one or more restriction enzymes, and the resulting fragments are
separated according to size by electrophoresis through an agarose gel. The DNA is then denatured into
single-stranded in situ and transferred from the gel to a membrane. The DNA attached to the membrane
is hybridized to radiolabeled DNA or RNA, and autoradiography is used to locate the positions of bands
complementary to the probe.

cDNAsynthesis
1. LinearizeRNA
TotalRNA
3ul
10mMdNTP
1ul
3Primer(50pmoles/ul)
0.5ul
DEPCtreatedH2O
3.5ul
Total

8ul
Incubatethesampleat65Cfor5min,thenplaceonice.

2. Prepareamastermixture

5XRTbuffer
4ul
25mMMgCl2
4ul
0.1MDTT
2ul
RNaseOUT
1ul
SuperScriptII
1ul
Total

12ul

3. Synthesis
MixmastermixturewithlinearizedRNA,thengoto
42C
60min
72C
15min
Chillonice.
BriefCF.Add1ulRNaseH,thenincubateat37Cfor20min.

PurificationofHistaggedproteinexpressedfrominsectcells
(Undernativeconditions)
1.
2.
3.
4.
5.
6.
7.

8.
9.
10.
11.

Cellswereharvested4872hpostinoculation(with5MOI)byCF(2000gfor5
min).
FlashfreezethepelletinliquidN,thenresuspendthecellsinlysisbuffer(1mlfor6
or12wellplate;5mlfor25cm2flask;10mlfor75cm2flask).
Cellswerelysedbythreecyclesoffreezingandthawing(liquidN37C).
Usinga20gaugedoublelockneedleand2syringes,shearthesamplebypassage
throughtheneedle34times.
CelldebriswaspelletedbyCFat8,000gfor10min(save50ulsupernatantfor
sample1;partofpelletforsample2).
Addnickelresin(equilibratedwithwashingbuffer;use100ulfor1mllysisbuffer,
250ul5ml,500ul10ml)intotheclearedlysate.
Loadthelysatewithresinonanemptycolumnwiththeoutletclosed.Aftertheresin
settleddown,opentheoutletandcollecttheflowthrough.Lettheflowthroughpass
thecolumnonemoretime(collect50ulforsample3).
Washtheresinwithwashingbuffer(3volumeofthelysate,sample4).
Elutetheproteinwithelutionbuffer(1/5volumeofthelysisbuffer,collect25ulfor
sample5).
Eluteoncemore(collect25ulforsample6).
Concentratethecombinedelutetoabout1mlwithCentricon30at6000rpm(sample
7).

Note:Trytokeepsamplesat4Coronicetopreventproteolysis.
Cellsalsocanbelysedbyadding0.1%TritonX100intolysisbuffer.Vortexcellsinthe
lysisbuffertosuspendandbreakthecells.Incubatethesamplesonicefor3045min(vortexat
10minintervalstoassistlysis).
Buffers:
Lysisbuffer

50mMNaH2PO4,300mMNaCl,5mMimidazole,0.05%Tween20,pH
8.0,addproteaseinhibitorbeforeuse(10ul/ml)
Washingbuffer
50mMNaH2PO4,300mMNaCl,5mMimidazole,0.05%Tween20,pH
8.0
Elutionbuffer

50mMNaH2PO4,300mMNaCl,200mMimidazole,0.05%Tween20,
pH8.0

Purificationofnativeproteinexpressedfrominsectcells
(UndernativeconditionswithDEAEcolumn)

1. Cellswereharvested4872hpostinoculation(with5MOI)byCF(2000gfor5min).
2. FlashfreezethepelletinliquidN,thenresuspendthecellsinlysisbuffer(1mlfor6or
12wellplate;4mlfor25cm2flask;10mlfor75cm2flask;20mlfor225cm2flask).
3. Cellswerelysedbythreecyclesoffreeze(liquidN2)andthaw(37C).
4. Usinga20gaugedoublelockneedleand2syringes,shearthesamplebypassage
throughtheneedle34times.
5. CelldebriswaspelletedbyCFat8,000gfor10min(save20ulsupernatantforsample
1;partofpelletforsample2).
6. 10mloftheclearedlysatewasloadontoaDEAESepharoseFastFlowcolumnataflow
rateof0.5ml/min.Afterloadingthesample,runfor10minwithcarryingbuffer(20mM
Tris,pH8.0plus50mMNaCl)onlywithoutcollecting.
7. ElutetheproteinwithTris,NaClsolution(20mlofeachoffollowingNaCl:200mM
250mM300mM350mM400mM500mM)ataflowrateof0.5ml/min.
Collectafractionforevery10min(4fractions/NaOHconcentration).Take6ulto
measureOD260andtake25ulsampletoprepareforWestern(add25ulof2XPAGE
samplebuffer,boilfor10min,load10ul/lane).
8. CombinethefractionswiththeproteinandloadontoCentricon30.CFat6000gat4C
tothefinalvolumeof1ml.
Note:Trytokeepsamplesat4Coronicetopreventproteolysis.
Cellsalsocanbelysedbyadding0.1%TritonX100intolysisbuffer.Vortexcellsinthe
lysisbuffertosuspendandbreakthecells.Incubatethesamplesonicefor3045min(vortexat
10minintervalstoassistlysis).

Buffers:
Lysisbuffer
20mMTris,20mMNaCl,pH8.0,addproteaseinhibitorCocktailbefore
use(5ul/ml,HaltTMProteaseInhibitorCocktailEDTAFree,Pierce)
Elutionbuffer

20mMTris,pH8.0plusdifferentconcentrationofNaCl.Prepare25ml
ofeachanduse~20ml.

Reference:
1. Mavrakisetal.2003.IsolationandcharacterizationoftheRabiesvirusNoP
complexproducedininsectcells.Virology305:406414.
2. Guptaetal.2003.Identificationofanoveltripartitecomplexinvolvedinreplication
ofVesicularstomatitisgenomeRNA.J.ofVirol.77:732738.

PurificationofHistaggedproteinexpressedfrombacteria
(Undernativeconditions)
1. Transferasinglecolony(besurefromanexpressionstrain)toaculturetubecontaining4
mlofmediumwithantibiotics,andgrowo/nat37Conashaker.
2. Inoculate2flasksofmedium(withantibiotics)withtheo/nculture(add2mlofculture
into30mlofmedium).Growthebacteriafor5060min(OD=0.50.7)at37Cwith
vigorousshaking(~2h).Growanotherflaskallthewayandpelletthecellsasuninduced
control.
3. AddIPTGtoafinalconcentrationof1mMintothecultureandgrowtheculturefor
another4h.Take1mlcultureandpelletthecellstochecktheproteinexpressionand
solubility,andpellettherestandstoreat20Cforpurification.
4. Resuspendthecellsin10mllysisbufferandsitonicefor20min.Cellswerelysedby
sonication(3X20secathighsetting)on,thenusinga20gaugedoublelockneedleand
2syringes,shearthesamplebypassagethroughtheneedle34times.
5. CelldebriswaspelletedbyCFat8,000gfor10min(save50ulsupernatantforsample
1;partofpelletforsample2).
6. Addnickelresin(equilibratedwithwashingbuffer;use100ulfor1mllysisbuffer,500
ul10ml,750ul15ml)intotheclearedlysateandmixgentlyfor30minonashaker.
7. CF2minat4000rpmanddiscardthesupernatant(sample3unbound).
8. Washtheresintwicewith10mlofwashingbufferbyCF.
9. Loadthelysatewithresinonanemptycolumnwiththeoutletclosed.Aftertheresin
settleddown,opentheoutletanddraintheflowthrough(collect50ulforsample4
wash).
10. Elutetheproteinwithelutionbuffer(1/5volumeofthelysisbuffer,collect25ulfor
sample5).
11. Eluteoncemore(collect25ulforsample6).
12. Concentratethecombinedelutetoabout1mlwithCentricon30at6000rpm(sample7).

Note:Trytokeepsamplesat4Coronicetopreventproteolysis.

Lysisbuffer
50mMNaH2PO4,300mMNaCl,0.05%Tween20,0.1%TritonX100,
pH8.0,plus100ug/mllysozyme(madefresh)
Washingbuffer
50mMNaH2PO4,300mMNaCl,5mMimidazole,0.05%Tween20,pH
8.0
Elutionbuffer
50mMNaH2PO4,300mMNaCl,200mMimidazole,0.05%Tween20,
pH8.0

PurificationofHistaggedproteinexpressedfrombacteria
(UndernativeconditionswithBugBusterfromNovagen)
1.
2.
3.
4.
5.
6.
7.

8.
9.
10.

Resuspendthecells(from40mlculture)in5mlofBugBuster.
Usinga20gaugedoublelockneedleand2syringes,shearthesamplebypassage
throughtheneedle34times.
CelldebriswaspelletedbyCFat8,000gfor10min(save50ulsupernatantfor
sample1;partofpelletforsample2).
Add1mlofnickelresin(equilibratedwithwashingbuffer)intotheclearedlysate
andmixgentlyfor30minonashaker.
CF2minat4000rpmanddiscardthesupernatant(sample3unbound).
Washtheresintwicewith10mlofwashingbufferbyCF.
Loadthelysatewithresinonanemptycolumnwiththeoutletclosed.Aftertheresin
settleddown,opentheoutletanddraintheflowthrough(collect50ulforsample4
wash).
Elutetheproteinwith2mlofelutionbuffer(collect25ulforsample5).
Eluteoncemore(collect25ulforsample6).
Concentratethecombinedelutetoabout1mlwithCentricon30at6000rpm(sample
7).

Note:Trytokeepsamplesat4Coronicetopreventproteolysis.


Washingbuffer
50mMNaH2PO4,300mMNaCl,5mMimidazole,0.05%Tween20,pH
8.0
Elutionbuffer
50mMNaH2PO4,300mMNaCl,200mMimidazole,0.05%Tween20,
pH8.0

PurificationofHistaggedproteinexpressedfrombacteria
(Underdenaturedconditions)

1. Transferasinglecolony(besurefromanexpressionstrain)toaculturetubecontaining4
mlofmediumwithantibiotics,andgrowo/nat37Conashaker.
2. Inoculate2flasksofmedium(withantibiotics)withtheo/nculture(add2mlofculture
into40mlofmedium).Growthebacteriafor5060min(OD=0.50.7)at37Cwith
vigorousshaking.Growanotherflaskallthewayandpelletthecellsasuninduced
control.
3. AddIPTGtoafinalconcentrationof1mMintothecultureandgrowtheculturefor
another4h.Take1mlcultureandpelletthecellstochecktheproteinexpression,and
pellettherestandstoreat20Cforpurification.
4. Resuspendthecellsin10mllysisbuffer.Cellswerelysedbysonication(3X30secat
highsettingandtakecaretoavoidfrothing).Thelysateshouldbetranslucentwhenlysis
iscomplete.Usinga20gaugedoublelockneedleand2syringes,shearthesampleby
passagethroughtheneedle34times.
5. CelldebriswaspelletedbyCFat12,000gfor10min(save50ulsupernatantforsample
1;partofpelletforsample2).
6. Add500ulofnickelresin(equilibratedwithwashingbuffer)intotheclearedlysateand
mixgentlyfor30minonashaker.

7. CF2minat4000rpmanddiscardthesupernatant(sample3unbound).
8. Washtheresintwicewith10mlofwashingbufferbyCF.
9. Loadthelysatewithresinonanemptycolumnwiththeoutletclosed.Aftertheresin
settleddown,opentheoutletanddraintheflowthrough(collect50ulforsample4
wash).
10. Elutetheproteinwithelutionbuffer(1/5volumeofthelysisbuffer,collect50ulfor
sample5).
11. Eluteoncemore(collect50ulforsample6).
12. Add2mlH2Otocombinedeluteandconcentrateittoabout1mlwithCentricon30at
6000rpmat10C(take50ulforsample7).
Buffers:
Lysisbuffer

100mMNaH2PO4,10mMTrisCl,8Murea,pH8.0
Washingbuffer
100mMNaH2PO4,10mMTrisCl,6Murea,pH6.3
Elutionbuffer
100mMNaH2PO4,10mMTrisCl,6Murea,pH4.5
Note:Duetothedissociationofurea,thepHoftheabovebuffersshouldbeadjusted
prioreachpurification.Donotautoclave.

Checktheproteinexpressionfrombacteria
1. Transferasinglecolony(besurefromanexpressionstrain)toaculturetubecontaining2mlof
mediumwithantibiotics,andgrowo/nat37Conashaker.
2. Inoculate2tubescontaining4mlofmediumwiththeo/nculture(200ulofculture/tube).Grow
thebacteriafor4050min(take1mltocheckOD=0.50.7)at37Cwithvigorousshaking.
3. AddIPTGtoafinalconcentrationof1mMintoonetubeandgrowthecultureforanother3h.
Leavetheothertubeasuninducedcontrolontheshaker.
4. Harvestcellsfrom1.0mlculturebyCF,thenresuspendthepelletin200ulofSDSPAGE
samplebuffer.Boilthesamplefor10min,thenCFfor10minandload10ulofthesupernatant
ontothegel.Useuninducedascomparison.
5. AfterCoomassiestaining,comparethebandpatternforproteinexpression(totalprotein).
6. Harvestcellsfromanother1.0mlculturebyCF,thenfreezethecellpelletforcheckingthe
proteinsolubility.

Note:
A,

Wetransformnormal(ex.Top10)cellswithourconstructfirst.Afterconfirmingthe
rightconstruct,itneedstobetransformedintoanexpressionE.colistrain(suchasBL21
orRosetta).

B,

IfuseRosettacells,LBshouldcontain35ug/mlofchloramphemicolplusanother
antibioticswhichyourplasmidresistantto.

C,

Solubilitytests:Resuspendthefrozencellpelletin200ulofBugBuster(Novagen).
Incubatefor10minatRTwithshaking,thenCFfor5min.Supernatantshouldbe
solubleandpelletshouldbeinsoluble.AddSDSPAGEsamplebuffer(1:1to
supernatantand100ultopellet)andload10ul/lanetorunthegel.

PurificationofPbypassingthroughSephacrylcolumn
Columnlength:27cm(afterpacking)
Columndiameter:1cm(inside)
Elutionspeed:0.4ml/min(pumpsetting:04)
Sampleamount:1ml(2.5minat0.4ml/min)
Fraction:5min/fraction(startcollectingafterloadingthesample)
Elutionbuffer:20mMTris,pH8.0

ProteinBlottingProteinOverlay(FarWestern):
RunSDSPAGE
Transferproteinontomembrane
Proteinoverlayanddetection
1.
2.

Aftertransfer,membranewaswashedwithH2O(20minonshakeratRT)toremove
SDS.
Themembranewasthenincubatedovernightat4ConashakerwithTBScontaining5%
skimmeddrymilktorenaturingtransferredproteinsandblockingnonspecificbinding
sitesonthemembrane.AllotherincubationandwashingweredoneatRTonashaker
andincubationbuffer(exceptsubstratebuffer)contains5%milk.

3.

Themembranewascutintostripsandincubatedwithcelllysate(1/4inTBS)expressing
baitproteinfor3h.
Wash3X10mininH2O.
IncubatethemembranewithprimaryantibodyagainstbaitproteininTBSfor2h.
Wash3X10mininH2O.
IncubatethemembranewithsecondaryAb(makesureuserightAbatproperdilution)in
TBSfor2h.
Wash3X10mininH2O.
DevelopinNBTBCIPdyesolution(coverfromlightforabout10min).
Rinsewithwaterandairdrythemembrane.

4.
5.
6.
7.
8.
9.
10.

Celllysis:
1.

2.

Forinsectcell:Resuspendthecells(T75flask)in15mloflysisbuffer(20mMTris,
20mMNaCl,pH8.0containing0.1%TritonX100).Usinga20gaugedoublelock
needleand2syringes,shearthesamplebypassagethroughtheneedle34times.
Add15mlofglycerol,mixwellandstoreat20C.
ForE.colicell:Resuspendthecells(40mlculture)in15mlofTBSplus0.1%Triton
X100).Lysethecellsbysonication(3X30sec).Add15mlofglycerol,mixwell
andstoreat20C.

PhosphoproteinGelStaining
1. Runtwoidenticalgels(normalSDSPAGE).
2. Fixthegelin50mlfixerovernight.
3. WashthegelinH2O(10minX3times).Afterwards,onegelforphosphoprotein
staining(ProQDiamondPhosphoproteinGelStaining),andtheotherfortotalprotein
staining(SYPRORubyProteinGelStaining).

PhosphoproteinGelStaining
1. Stainthegelinthedarkin50mlofProQstainingsolutionfor75min.
2. Destainthegelinthedarkin90mlofDestainsolution(30minX3).

3. WashthegelwithH2O(5minX2).
4. Viewthebands(SetbottomboxONandSYBRGoldCY3orEB).
5. Gelbandsfadeafter23h.

TotalProteinGelStaining
a. Stainthegelinthedarkin50mlofSYPRORubystainingsolutionfor5hor
longer.
b. Destainthegelinthedarkin90mlofDestainsolution(30minX2).
c. TransferthegelintoH2O(bandsdonotfadefordays).
d. Viewthebands(SetbottomboxONandEBorSYBRGoldCY3).
Note:Performallfixation,stainingandwashingstepsonanorbitalshakerat50rpminalarge
plasticweighingdish.
Solutions:
Fixer
50%methanol
10%aceticacid
Destain(fortotalprotein)
10%ethanolormethanol
7%aceticacid

Leaderbindinggelshiftassay
PCR:
Designapairofprimers(5withT7promoterplusacloningsiteXbaI;3withabluntend
enzymesiteSnaBIplusacloningsiteHindIII).

CloneintopUC19(nopromoter):

CloneintopUC19atXbaIandHindIII.Domini+midiprep.

Linearizethetemplate:

plasmidDNA(25ug)
buffer4
enzyme
H2O

20

ul

10
ul
2
ul
68
ul

Total

100

ul
Incubatefor3hat37C.

DophenolChloroformextraction;ethanolprecipitation,and
resuspendin20ulofH2Othenuse10ultodotranscription.

RNAtranscription:
Epicentrekit
KeepallcomponentsoniceandcombinethereactionmixtureatRTinthefollowingorder.
Cold:
Hot
H2O
2.8ul
0.8ul
Linearizedtemplate(1ug)
10ul
10ul
10XAmpliScribeT7/T3ReactionBuffer
2ul
2ul
100mMATP
0.75ul
0.5ul
100mMCTP
0.75ul
0.5ul
100mMGTP
0.75ul
0.5ul
100mMUTP
0.75ul
1mM 0.5ul
32
PUTP

2ul
RNaseinhibitor(RNAguard)
0.2ul
0.2ul
100mMDTT
2ul
2ul
AmpliScribeT7enzyme
1ul
1ul
total20ul
Incubatefor2hat37C,thencleanup(phenolextraction..).Forcoldtest,take5ul
(beforecleanup)andrungel(150V15min).

Cleanup:
1.
2.
3.
4.
5.
6.

Add90ulH2O,50ulH2Osaturatedphenol,50ulchloroform.
Vortexthoroughly,thenCFfor5min.
Pipette95ulaqueousphase(top)intoanewtube
Add300ulIsopropanol:10MNH4Ac(10:1)solutionandmixwell.
Incubatefor30minorlongerat20C.
CFfor15minatmaxat4C.

7.
8.
9.
10.
11.

Pouroffsolution,andadd700ul70%ethanol
CFfor10minatmaxat4C.
Pouroffethanolthoroughly
Airdrythepelletfor1520min
Dissolvethepelletin200ulof1XTEadd0.5ulRNaseinhibitor.Aliquotto40
ulandstoreat20C.

Gelshiftassay:

Bindingreaction:
BindingBuffer
4.8ul
RNAguard
0.2ul
tRNA(0.1mg/ml)
1ul
Protein(inglycerol)
2ul
32
PlabeledRNA
2ul
Incubatefor15minatRT.Add2ulloadingbuffer,loadallontothegelandrunnon
denaturingPAGEwith1XTAEfor1hat4C.Aftertherun,transferthegelonto3Mfilter
paper,coverwithaplasticsheetandvacuumdryit.
ChecktheradioactivitywithaGeigercounterandexposedtothecleanedPhosphor
screenfor3ho/n.
5%PAGEnondenaturinggel(10mlfor2smallgels):
H20
50XTAEbuffer
30%acrylamide/bis(37.5:1)
10%APS
TEMED

8.00ml
200ul
1.67ml
100ul
8ul

Bindingbuffer:200mMNaPO4,pH7.5containing600mMNaCl.
Saran wrap
gel
3MM paper
2 sheets of paper

Gel dryer bed

dsRNAtranscription(followStratagenesDicerenzyme):
A.

DesignPCRprimerswithT7promoterinbothprimersandrunPCRtoproducethe
transcriptiontemplate.

B.
C.
D.

CheckthePCRconcentrationanduse1ugtodotranscription.
UseEpicentrekittodotranscription(makeupto20ulwithH2O).
CleanupasRNAtranscription.

DNAbindingassay:
A. DesignPCRprimerswithoutT7promoterinbothprimersandruncoldPCR.
B. CheckthecoldPCRproductbyrunningthegel.
C. RunhotPCR(1ulof10mMdATP,dGTP,dTTPand0.1mMdCTPplus5ulP32
dCTP).
D. CleanupasDNA(add50ulH2O,50ulphenol,50ulchloroform,CF5thendoEtOH
precipitationandresuspendin200ulH2O).

Northwesternblot(Zehneretal.,1997.NucleicAcidsRes.25:3362)
RunSDSPAGE
Transfertonitrocellulosemembrane
Bindingassay
1.
2.
3.
4.
5.
6.

Aftertransfer,membranewaswashedwithH2O(2X10minonshakeratRT)toremove
SDS.
Themembranewasthenincubatedovernightat4ConashakerwithTBScontaining5%
skimmeddrymilktorenaturingtransferredproteinsandblockingnonspecificbinding
sitesonthemembrane.AllotherincubationandwashingweredoneatRT.
Wash2X10mininH2O.
Incubatethemembranewith32PlabeledRNAprobeagainstbaitproteininbindingbuffer
for2h.
Wash3X10mininbindingbuffer.
Drythemembrane,checktheradioactivitywithaGeigercounterandexposedtothe
cleanedPhosphorscreenfor3ho/n.

Bindingbuffer:
50mM TrisHClpH7.5,50mM NaCl,0.1mMEDTA,1mMDTT,5U/ml
RNasin

NICOTIANA BENTHAMIANA CALLUS CULTURE

Note: Work under aseptic conditions! Sterilize all solutions and accessories prior to using
them!

A. Germination of N. benthamiana seeds under aseptic conditions

Wash down hood thoroughly with 10% bleach solution before starting!
Surface sterilize Nicotiana benthamiana seeds:
1. Fill up two sterile graduated 1.5 ml eppendorf tube to ~ 50 l line with N.
benthamiana seeds.
2. Add 700 l 70% ethanol, close tube and shake it several times. Wait a few seconds
for seeds to settle down on the bottom of the tube. Remove (discard) ethanol with
a sterile 1 ml pipet tip (use new tip for each tube)
3. Repeat the above step 4 more times.
4. Add 700 l undiluted Clorox bleach and 35 l 10% SDS. Shake tube up and down
for 9-10 min. Wait for seeds to settle down. Remove bleach solution (use new tip
for each tube).
5. Add 700 l sterile dH2O. Invert tube a few times, wait for seeds to settle down, then
remove dH2O. Every time use a fresh tip for each tube. Repeat this step 4 more
times (5 rinses total). Finally, add 350 l dH2O to seeds in the tube. Use a sterile
large orifice 1 ml tip to place seeds on two MS-E plates (1 plate for each tube).
Spread/separate seeds as much as possible using sterile bacterial loops. Do not dry
plates after spreading seeds.
Seal plates with parafilm. Incubate plates at 23 C o, 16 h light and 8 h dark.

B. Induction of N. benthamiana callus culture

Use MS-E plates for N. benthamiana. Use 100X20mm petri plates. Can store plates for
several weeks at 4C.

First passage can be made 3-5 weeks after plating. Cut leaves with a sterile scissors,
induce wounds by squeezing leaves w/sterile forceps and pressing gently onto medium
(MS-E).
Seal plates with parafilm. Incubate plates at 23 C o, 16 h light and 8 h dark.

C. Maintainance of N. benthamiana callus culture on MS

Use MS plates for N. benthamiana callus maintenance. Let the plates dry under the
hood with the lid partially covering a dish. Longer storage at 4C is okay.
From this point on only MS medium is used.
Callus should be light yellow with a little bit of green. Brown color indicates dead or
dying tissue. Good callus is friable but not too soft. Transfer only healthy looking callus!
Do not passage callus which is too soft or brownish. 1 plate will make 2-3 new plates by
dividing callus into ~ 3 mm pieces with forceps. Transfer ~ 20 such pieces into a fresh
plate. Distribute pieces evenly on the plate.
Germination: 3 weeks (seeds plated on MS-E, 2 plates)
1st passage: 4 weeks (transfer seedlings on MS-E to MS, usually 5-6 plates) = 1P
2nd passage: 3 weeks = 2P (usually 5-6 plates)
3rd passage: 2 weeks = 3P
4th passage: 2 weeks = 4P
5th passage: 2 weeks = 5P
Seal plates with parafilm. Incubate plates at 23 C o, 16 h light and 8 h dark.
The 3rd, & 4th and 5th (though 5th seems to be less valuable) ) passages seem to be
best for protoplast isolation (Zivile).

Primerdesign
Size:2225bp(orlonger).Sizewilldeterminethemeltingtemperature(Tm).
Tm=2x(A+T)+4x(C+G);NormallyTmshouldbehigherthan55C.

Iftheprimeristooshort,Tmwillbeverylow,andthespecificitywillbereduced.
WhencalculatingTm,donotcountthemodifiedbpsplus23bpsof5protectingends.
Sequence:The3endoftheprimeristhemostimportantpartfortheamplification.Itmustbe
strictlycomplementarytothetemplatesequence,andifpossible,haveahighG+Ccontent.
Sequencemodification(restrictionsite,mutationsite,sitedirectedmutagenesis):Alwayskeepa
complementary 3 end and try to introduce the modification site at the 5 end (leave 23
complementarybptoprotectthesite).Forsitedirectedmutagenesis,introducethemodification
siteinthemiddleofbothprimers.
Specialrules:
A,
Ifyouaregivenasequence(singlestrandDNA),thissequenceis(+)sequence.
The3primershouldbecomplementarytothesequence;andthe5primershouldbethe
samesequenceasthetemplate(thegivensequence).
Forexample:
Givensequence
5CATGCCTTAAGCCCG..................................... TTCCATGGAACTGTT3
5CTTAAGCCCG3
3GGTACCTTGACAA5
5primer
3primer
B,
Agivenvirussequence(singlestrandDNA)isthesamesequenceasmRNA(with
UreplacedbyT).Forexample:Todesigna5primerwithastartcodon(5AUG3)and
a3primerwithastopcodon(5UGA3):

Givensequence
5CATGCCTTAAGATACGTC....................CGTCATTCCATGGACTTGTG3
5CAATGCTTAAGATACGTC3...........3GCAGTAAGGTACCTACTCAC5
5primer
3primer
C,
Ifyouneedtoaddenzymestoyourprimer,followenzyme5to3.Forexample:
To design a 5 primer with BamH I (5GGATCC3) and a 3 primer with AvrII
(5CCTAGG3):
Givensequence
5GCTTAAGATACGTC.............CGTCATTCCATGGGTG3
5GGATCCGCTTAAGATACGTC3....3GCAGTAAGGTACCACGGATCC5
5primer
3primer
Ourlabuse0.5ulof50pmoles/ultodoPCR.Ifproblemhappenedandprimersaresuspected,
load5ulof50pmoles/ulontogelandrunfor10minat120Vtocheck.
dsRNAtranscriptionandconfirmation(followDicerEnzymeManualfromStratagene)

ToconfirmdsRNA:
2
1.5
or 1.5
1
5.5

uldsRNAtranscriptionproduct
ul2MNaCl(final0.3M)
ul0.2MNaCl(final0.03M)
ul0.5mg/mlRNaseA
ulTE

37Cfor1h,thenrunthegel.dsRNAwillbedigestedbyRNaseAunderlowsalt
condition,butnothighsaltcondition.

PurificationofHistaggedproteinexpressedfromplant
(Undernativeconditions)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Leaveswereharvested34dayspostinfiltration.
Leavesweregroundwithachilledmortarandpestleinextractionbuffer
(leaf:buffer=1:5).
CFthehomogenatefor15minat10,000rpmat4C.FilterthroughMiracloth
(take50ulsupernatantandaddinto25ulsamplebufferassample1.)
Add250ulnickelresin(equilibratedwithextractionbuffer)intothesupernatant
(6mlHistagsample+6mlnonetagsample).
Mixfor30min1honashakeronice.
CF2minat4000rpmanddiscardthesupernatant.
Washtheresintwicewith10mlofwashingbufferbyCF.Transferresinwith1
mlwashingbufferintoaneppendorf,spinandremovethesupernatant.
Add500elutionbufferintoresinandincubatefor10min.
CFandcollectsupernatant(50ul+50ulSBassample2).
Take50ulresinaddto50ulSBassample3.

Extractionbuffer
300mMNaH2PO4,0.1%BME,pH7.5

Washingbuffer
100mMNaH2PO4,100mMNaCl,5mMimidazole,pH7.5
Elutionbuffer
50mMNaH2PO4,100mMNaCl,200mMimidazole,pH7.5

RNAPurificationwithRNAzolRT(MolecularResearchCenter)
(AllCFisdoneatmaxspeedatRmT)

1.
2.
3.
4
5a
6a
7a
8a
9a
5b
6b
7b

Grind70100mgleafinliquidnitrogenwithamortarandpestle.
Add1mlofRNAzolRTreagent(Cat#:RN190)andgrindmore.Add400ul
H2O,mixwell,transferintoa1.5mltubeandsitfor15minatRmT.
CFthehomogenatefor15min.
Transfer1.1mlofthesupernatanttoafreshtube(goto5aor5bfromhere).
Add440ulof75%ethanol,mixwellandsitfor5min.
CF10min.(Save800ulsupernatantforsmallRNAisolation)
Washthepellettwicewith400ulof75%ethanol(CFfor2mineachtime).
RemoveallleftoverethanolanddrythepelletatRmTfor15min.
DissolvetheRNAin50ulofH2O(repeatedpipetting).
Add60ulchloroformtothesupernatant(fromstep4),vertexfor15secandCF
10min.
Transfer800ulofaqueousphase(nocolor)intoanewtube(ifyouwanttotal
RNA,goto5c)
Add320ulof75%ethanol,mixwellandsitfor5min.

8b

Dothesameas6athrough9a

5c

Add640ulofisopropanoltotheaqueousphase(fromstep6b),mixwellandsit
for10min.
CF10min.
Dothesameas7athrough9a

6c
7c

SmallRNAisolation
1. Add640ulofisopropanolto800ulsupernatantsavedfor6a,mixwellandstorefor30
minat4C.
2. CF15min.
3. Washthepellettwicewith400ulof70%isopropanol.
4. RemoveallleftoverisopropanolanddrythepelletatRmTfor15min.
5. DissolvetheRNAin50ulofH2O(repeatedpipetting).