all chapter 3.docx

all chapter 3.docx

《all chapter 3.docx》由会员分享，可在线阅读，更多相关《all chapter 3.docx（60页珍藏版）》请在冰点文库上搜索。

allchapter3

Chapter3WorkingwithBacteriophageM13Vectors

Protocol1:

PlatingBacteriophageM13

BacteriophageM13formsturbidplaquesonlawnsofmalestrainsofE.coli.

Protocol2:

GrowingBacteriophageM13inLiquidCulture

MostmanipulationswithM13,includingpreparationsofviralstocksandisolationofsingle-anddouble-strandedDNAs,beginwithsmall-scaleliquidculturesthatareinfectedwithanM13plaque,pickedfromanagarplate.

Protocol3:

PreparationofDouble-stranded（ReplicativeForm）BacteriophageM13DNA

Thedouble-strandedreplicativeform（RF）ofbacteriophageM13isisolatedfrominfectedcellsusingmethodssimilartothoseusedtopurifyplasmidDNA.SeveralmicrogramsofRFDNAcanbeisolatedfroma1-2-mlcultureofinfectedcells.

Protocol4:

PreparationofSingle-strandedBacteriophageM13DNA

BacteriophageM13single-strandedDNAispreparedfromvirusparticlessecretedbyinfectedcellsintothesurroundingmedium.Thefilamentousparticlesareconcentratedbyprecipitationfromahigh-ionic-strengthbufferwithpolyethyleneglycol.Subsequentextractionwithphenolreleasesthesingle-strandedDNA,whichisthencollectedbyprecipitationwithethanol.Thisprotocolisgenerallyusedtopreparesingle-strandedDNAfromasmallnumberofM13isolates.Typically,theyieldofsingle-strandedDNAis5-10µg/mlinfectedculture.

Protocol5:

Large-scalePreparationofSingle-strandedandDouble-strandedBacteriophageM13DNA

Thisprotocol,ascaled-upversionofChapter3,Protocol3andChapter3,Protocol4,isusedchieflytogeneratelargestocksofdouble-strandedDNAofstrainsofM13thatareroutinelyusedascloningvectors.Largeamountsofsingle-strandedDNAofanindividualrecombinantmayoccasionallybeneededforspecificpurposes,e.g.,togeneratemanypreparationsofaparticularradiolabeledprobeortoconstructlargenumbersofsite-directedmutants.

Protocol6:

CloningintoBacteriophageM13Vectors

ThisprotocoldescribesthreestandardmethodstoconstructbacteriophageM13recombinants:

（1）ligatinginsertDNAtoalinearizedvector,preparedbycleavageofM13RFwithasinglerestrictionenzyme;

（2）usingalkalinephosphatasetosuppressself-ligationofthelinearizedvector,and（3）usingM13RFcleavedwithtworestrictionenzymesfordirectionalcloning.

Protocol7:

AnalysisofRecombinantBacteriophageM13Clones

Arapidmethodtoanalyzethesizeofthesingle-strandedDNAofM13recombinants.

Protocol8:

ProducingSingle-strandedDNAwithPhagemidVectors

Thisprotocoldescribesmethodstosuperinfectbacteriacarryingarecombinantphagemidwithahigh-titerstockofanappropriatehelpervirusandtoassaytheyieldoffilamentousvirusparticlesthatcarrysingle-strandedcopiesofthephagemidDNA.Thekeytosuccessinusingphagemidsistoprepareastockofhelperviruswhosetiterisaccuratelyknown.

Chapter3,Protocol1

PlatingBacteriophageM13

BacteriophageM13formsturbidplaquesonlawnsofmalestrainsofE.coli.

CAUTION

RECIPE

MATERIALS

BuffersandSolutions

IPTG（20%w/v）

X-galsolution（2%w/v）

Media

LBagarplatescontainingtetracyclineorkanamycin

Theseplatesareneededonlyifatetracycline-resistantstrainofE.coli,suchasXL1-Blue,orakanamycin-resistantstrainofE.coli,suchasXL1-BlueMRF´Kan,isusedtopropagatethevirus.

M9minimalagarplates,supplemented

TheseplatesareneededwhenusingE.colistrainsthatcarryadeletionoftheprolinebiosyntheticoperon（[lac-proAB]）inthebacterialchromosomeandthecomplementingproABgenesontheF´plasmid.

RichM13medium

RichM13agarmediumplatescontaining5mMMgCl2

RichM13topagaroragarosecontaining5mMMgCl2

VectorsandBacterialStrains

BacteriophageM13stock

LBorYTmediumfromafullygrownliquidcultureofbacteriainfectedwithbacteriophageM13containsbetween1010and1012pfu/ml.AbacteriophageM13plaquecontainsbetween106and108pfu.

E.coliF´strain,preparedasamasterculture

METHOD

1.StreakamastercultureofabacterialstraincarryinganF´plasmidontoeitherasupplementedminimal（M9）agarplateoranLBplatecontainingtetracycline（XL1-Blue）orkanamycin（XL1-BlueMRF´Kan）.Incubatetheplatefor24-36hoursat37°C.

2.Toprepareplatingbacteria,inoculate5mlofLBorYTmediumina20-mlsterileculturetubewithasingle,well-isolatedcolonypickedfromtheagarplatepreparedinStep1.Agitatetheculturefor6-8hoursat37°Cinarotaryshaker.Chillthecultureinanicebathfor20minutesandthenstoreitat4°C.Theseplatingbacteriacanbestoredforperiodsofupto1weekat4°C.

Donotgrowthecellstosaturation,asthiswillincreasetheriskoflosingthepiliencodedbytheF´plasmid.

3.Preparesteriletubes（13x100mmor17x100mm）containing3mlofmeltedLBorYTmediumtopagaroragarose,supplementedwith5mMMgCl2.Allowthetubestoequilibrateto47°Cinaheatingblockorwaterbath.

4.Labelaseriesofsteriletubes（13x100mmor17x100mm）accordingtothedilutionfactorandamountofbacteriophagestocktobeadded（pleaseseeStep5）,anddeliver100µlofplatingbacteriafromStep2intoeachofthesetubes.

5.Preparetenfoldserialdilutions（10-6to10-9）ofthebacteriophagestockinLBorYTmedium.Dispense10µlor100µlofeachdilutiontobeassayedintoasteriletubecontainingplatingbacteriafromStep4.Mixthebacteriophageparticleswiththebacterialculturebyvortexinggently.

6.Add40µlof2%X-galsolutionand4µlof20%IPTGsolutiontoeachofthetubescontainingtopagar.Immediatelypourthecontentsofoneofthesetubesintooneoftheinfectedcultures.Mixtheculturewiththeagar/agarosebygentlyvortexingfor3seconds,andthenpourthemixtureontoalabeledplatecontainingLBorYTagarmediumsupplementedwith5mMMgCl2andequilibratedtoroomtemperature.Swirltheplategentlytoensureanevendistributionofbacteriaandtopagar.

Workquicklysothatthetopagarspreadsovertheentiresurfaceoftheagarbeforeitsets.

7.RepeattheadditionoftopagarwithX-galandIPTGforeachtubeofinfectedculturepreparedinStep5.

8.Replacethelidsontheplatesandallowthetopagar/agarosetohardenfor5minutesatroomtemperature.WipeexcesscondensationoffthelidswithKimwipes.Inverttheplatesandincubatethemat37°C.

Paleblueplaquesbegintoappearafter4hours.Thecolorgraduallyintensifiesastheplaquesenlargeandiscompleteafter8-12hoursofincubation.

RECIPES

5xM9Salts

Na2HPO4•7H2O,64g

KH2PO4,15g

NaCl,2.5g

NH4Cl,5.0g

deionizedH2O,to1liter

Dividethesaltsolutioninto200-mlaliquotsandsterilizebyautoclavingfor15minutesat15psi（1.05kg/cm2）onliquidcycle.

CaCl2

Dissolve11gofCaCl2•6H2Oinafinalvolumeof20mlofdistilledH2O.Sterilizethe2.5Msolutionbypassingitthrougha0.22-µmfilter.Storein1-mlaliquotsat4°C.

IPTG

IPTGisisopropylthio--D-galactoside.Makea20%（w/v,0.8M）solutionofIPTGbydissolving2gofIPTGin8mlofdistilledH2O.Adjustthevolumeofthesolutionto10mlwithH2Oandsterilizebypassingitthrougha0.22-µmdisposablefilter.Dispensethesolutioninto1-mlaliquotsandstorethemat-20°C.

LB

deionizedH2O,to950ml

tryptone,10g

yeastextract,5g

NaCl,10g

Forsolidmedium,pleaseseeMediaContainingAgarorAgarose.

ToprepareLB（Luria-Bertanimedium）,shakeuntilthesoluteshavedissolved.AdjustthepHto7.0with5NNaOH（approx.0.2ml）.Adjustthevolumeofthesolutionto1literwithdeionizedH2O.Sterilizebyautoclavingfor20minutesat15psi（1.05kg/cm2）onliquidcycle.

M9

sterileH2O（cooledto50°Corless）,to750ml

5xM9salts,200ml

1MMgSO4,2ml

20%solutionoftheappropriatecarbonsource（e.g.,20%glucose）,20ml

1MCaCl2,0.1ml

steriledeionizedH2O,to980ml

Forsolidmedium,pleaseseeMediaContainingAgarorAgarose

Ifnecessary,supplementtheM9minimalmediumwithstocksolutionsoftheappropriateaminoacidsandvitamins.

PreparetheMgSO4andCaCl2solutionsseparately,sterilizebyautoclaving,andaddthesolutionsafterdilutingthe5xM9saltsto980mlwithsterileH2O.Sterilizetheglucosebypassingitthrougha0.22-µmfilterbeforeitisaddedtothedilutedM9salts.

WhenusingE.colistrainsthatcarryadeletionoftheprolinebiosyntheticoperon[（lac-proAB）]inthebacterialchromosomeandthecomplementingproABgenesontheF'plasmid,supplementtheM9minimalmediumwiththefollowing:

0.4%（w/v）glucose（dextrose）

5mMMgSO4•7H2O

0.01%thiamine

MediaContainingAgarorAgarose

Prepareliquidmediaaccordingtotherecipesgiven.Justbeforeautoclaving,addoneofthefollowing:

BactoAgar（forplates）

15g/liter

BactoAgar（fortopagar）

7g/liter

agarose（forplates）

15g/liter

agarose（fortopagarose）

7g/liter

Sterilizebyautoclavingfor20minutesat15psi（1.05kg/cm2）onliquidcycle.Whenthemediumisremovedfromtheautoclave,swirlitgentlytodistributethemeltedagaroragaroseevenlythroughoutthesolution.Becareful!

Thefluidmaybesuperheatedandmayboiloverwhenswirled.Allowthemediumtocoolto50-60°Cbeforeaddingthermolabilesubstances（e.g.,antibiotics）.Toavoidproducingairbubbles,mixthemediumbyswirling.Platescanthenbepoureddirectlyfromtheflask;allowapprox.30-35mlofmediumper90-mmplate.Toremovebubblesfrommediumintheplate,flamethesurfaceofthemediumwithaBunsenburnerbeforetheagaroragarosehardens.Setupacolorcode