Screening of Recombinant Clones.docx
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Screening of Recombinant Clones.docx
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ScreeningofRecombinantClones
Protocol:
ScreeningofRecombinantClones
I.Objectives
1.UnderstandtheprincipleofDNAcloningtechnique.
2.Designtheprotocoltoscreenthecoloniesforrecombinants.
3.Givepossibleresultsandtrytoexplainthem.
II.materialsandmethods
1.PlasmidDNApurification
1)Reagents
Reagents
Composition
Attention&Explanation
Solution1
50mmol/Lglucose
10mmol/LEDTA
25mMTris-HCl
pH8.0
RNasehasbeenadded.
GlucoseistoenhancethebuoyantEDTAisnotsolublewherepHislow.
Solution2
0.2mol/LNaOH
1%SDS
1.BacterialCellwalllysis
2.DenaturalizationofDNA
Solution3
60ml5mol/LKAc
11.5mlaceticacid
28.5mLH2O
ToneutralizetheenvironmentthatleadtorenaturationofplasmidDNA.
2)Apparatus
Pipettes
EPPENDORFtube
Centrifuge
ElectrophoresisApparatus
UVGelimaginginstruments
IceBox
2.TheenzymedigestionofplasmidDNA:
1)Reagents
Reagents
Composition
Attention&Explanation
NEBstandardmolecularmarker
1kbDNAladder
Tomarkthelevelofweight
Endonuclease1
EcoRI(μl)
Endonuclease2
XhoI(μl)
10×Hbuffer
500mMTris-HCl(pH7.5)100mMMgCl2
10mMDithiothreitol
1000mMNaCl
Toprovidetheenvironmentsuitabletodigestforbothenzymes
TBE/TAEbuffer(10×)
Tousedasrunningbuffer
2)Apparatus
Pipettes
EPPENDORFtube
3.Electrophoresisgel:
1)Reagents
Reagents
Composition
Attention&Explanation
EB
3,8-diamino-5-ethyl-6-phenylphenanthridiniumboromide10mg/ml
ToinsertintothebasepairsofDNAmolecule
EBisakindofmutagen
Loadingbuffer(3×)
0.25%Bromophenolblue
40%(W/V)sucroseor30%glycerol
Tousedasanindicator
ToincreasethedensityofDNAincaseofdiffusion
2)Apparatus:
Electrophoresisapparatus
Electrophoresistrough
Gelimagingsystem
1.Selectionoftherecombinantclonesandtheculturetargetcells
Itisrequiredtoselectonepositiveclone(whitestrain)andonenegativeclone(bluestrain)asacontrolgroup.TransfertheclonetoLB(withampicillin)withaseptictoothpicktipandcultureat37℃overnight.
2.PlasmidExtraction
1)Inoculateasinglecolonyoftransformedbacteriainto1.5–3mlofmediumcontainingappropriateantibiotics.Incubatethecultureovernightat37℃.
2)Pour1.5-3mlofthecultureintoanEppendorftube.Centrifugeat10000r/minfor1min.Removethesupernatant.Suspendthebacterialpelletin100ulSolutionI(GET)byvigorousvortex.
3)Add200μloffreshlypreparedAlkalinelysissolutionII(0.2mol/LNaOH+1%SDS)toeachbacterialsuspension.Closethetubetightlyandmixthecontentsbyinvertingthetuberapidlyfivetimes.Donotvortex!
Standthetubeonicefor5min.
4)Add150μlofice-coldsolutionIII.Closethetubeandinvertthetubeseveraltimes.Storethetubeonicefor3-5minutes.
5)Centrifugethelysateat10000r/minfor7minutesinamicrofuge.Transferthesupernatanttoanewtube.Add1ml100%ethanol.Centrifugeat12000r/minfor5minutes.Removethesupernatant.
6)Washthepelletwith0.5ml70%ethanol,centrifugeat12000r/minfor5minutesandremovethesupernatant.Standthetubeinaninvertedpositiononapapertoweltoallowallofthefluidtodrainawayatroomtemperature
7)Dissolvethenucleicacidsin30-50μlwaterorTE(pH8.0)containing20μg/mlDNase-freeRNaseA.Incubatethesolutionatroomtemperaturefor15-30min.(Sometimesphenol/chloroformextractionisnecessary.)
8)StoretheDNAsolutionat-20℃.
3.Enzymedigestionandgelelectrophoresis
1)Addthefollowingcomponentsinto6Eppendorftubes.
PlasmidDNA
10×HBuffer(μl)
EcoRI(μl)
XhoI(μl)
H2O(μl)
Total(μl)
1ml
2
0.5
0.5
To20.0
20.0
Theplasmidsinthe6tubesareaddedasfollows
No.
Plasmid
1-5
Extractedfromwhitecolonies
6
Extractedfrombluecolonies(Controlgroup)
2)Incubateat37○Cinwaterfor30min
3)Duringtheincubatingperiod,preparetheagarosegelforelectrophoresis.PourtheagarosegelsolutiononaPMMAplate.Placeitatroomtemperatureforabout30minutestoletitfreeze.ThenputitintheelectrophoresisapparatusandmakesurethatTAEbufferinundatethegel.
4)Afterenzymaticcuttingfinished,Add10μl(3X)loadingbuffertoeachtube.
5)Abstract15μlsolutionfromeachtubeandaddthesampletothetargetwellsintheagarosegel.
6)Add6μl1kbDNAladder(50ng/μl)totwocertainwells.
7)Electrophoresisatthevoltageof120VuntiltheBromophenolbluewasabout1cmawayfromtheothersideofthegel.
8)DyethegelwithEBsolutionfor10min.
Imagingandphotographingautomaticallyunderultravioletradiationwithwavelengthof310nm
III.Predictiontoresults.
Inthisexperiment,therewillbe3kindsofplasmids.
Positive:
1)plasmidswithtargetsequenceinsertion
2)plasmidswithothersequenceinsertion
Negative:
3)plasmidswithnosequenceinsertion
Accordingly,therewillbe3kindsofresultsshowninthegel.Thefirstkindofplasmidsiswhatwewant.
1)3kblinearDNAplus800bpsegment.(Targetinsertion)
It’sclearthatthetargetsequenceisbetweenthetworestrictionsitesandcanbereleasedbydigestionwiththeenzymes.Aftergel-electrophoresis,theseproductscanbeclearlyshownonthegel,illustratingtheidentityoftheclones.
2)Nocutting,singlecuttingsite,or3kblinearDNAplusasegmentshorterorlongerthan800bp.(Contaminantsegmentinsertion).(contaminantsegmentinsertion)
Whentheinsertedsegmentisn’tthetargetsequence,therestrictionsiteisunknown.Thus,theresultmayvary.
3)3kblinearDNAplusaveryshortsegment(Noinsertion).
Theshortsegmentexcisedbytherestrictionenzymesisbetweenthetwocloserestrictionsites.
【Homework】
1.Vectorsforprokaryotes:
1)
PlasmidName
ACS1
Source/Vendor
AddgenePlasmidRepository
PlasmidType
Bacterialexpression
PlasmidSize
4700
SequencingPrimer
pQE80sequencingvector
ProteinTags
His
BacterialResistance
Ampicillin
2)
PlasmidName
ColE
Source/Vendor
HanLimlab
PlasmidType
BacterialExpression
PlasmidSize
2000
SequencingPrimer
Amp-R,pBR322ori-F
SequencingPrimerSequence
ATAATACCGCGCCACATAGC,GGGAAACGCCTGGTATCTTT
BacterialResistance
Ampicillin
3)
PlasmidName
p15TV-L
Source/Vendor
ClinicalGenomicsCentre,UniversityHealthNetwork,Toronto
PlasmidType
BacterialExpression
Viral/Non-viral
Non-viral
Promoter
T7
ExpressionLevel
High
PlasmidSize
7746
SequencingPrimer
T7-Fwd
SequencingPrimerSequence
AATTAATACGACTCACTATAGGG
ProteinTags
His
BacterialResistance
Amp
4)
PlasmidName
pAcGFP1
Source/Vendor
Clontech
PlasmidType
Bacterial
Promoter
lac
PlasmidSize
3300
ProteinTags
GFP(Nterm)
BacterialResistance
Ampicillin
2.VectorsusedinMammaliancells:
1)
PlasmidName
pAcGFP1-C1
Source/Vendor
Clontech
PlasmidType
Mammalian
Viral/Non-viral
Nonviral
Stable/Transient
Stable(transfected)
Constitutive/Inducible
Constitutive
Promoter
CMV
PlasmidSize
4700
ProteinTags
GFP(Nterm)
BacterialResistance
Kanamycin
MammalianSelection
Neomycin
2)
PlasmidName
p079pPNT6
Source/Vendor
AddgenePlasmidRepository
PlasmidType
Mammalianexpression,Cre/Lox
PlasmidSize
7500
SequencingPrimer
M13R
BacterialResistance
Ampicillin
MammalianSelection
Neomycin
3)
PlasmidName
p3xFlag-CMV-7.1
Source/Vendor
Sigma
PlasmidType
Mammalian
PlasmidSize
4700
ProteinTags
3xFlag(Nterm)
BacterialResistance
Amp
3.Comparisonofprokaryoticandmammalianexpressionvectors
Thedifferencebetweenprokaryoticexpressionvectorsandmammalianvectorsareshowninthetablebelow.
Feature
Prokaryoticexpressionvectors
Mammalianexpressionvectors
Shine-Dalgarnosequence
Essential
None
Promoter
T7,trporAraC
CMV,SV40,EF1alpha
SourceofPromoter
Phagesandinherentpromoters
Viruses
Polyadenylationsite
None
Essential,usuallySV40polyAsite
Replicationorigin
Essential,usuallyderivedfrompBR322orthepUCorigin
Onlyessentialforshuttlevectors,usuallypUCorigin
Antibiotics-Resistance
Essentialforscreening
Essentialforscreening,mayhavemulti-resistanceforshuttling
Enhancers
None
Recommendedforhigh-levelexpression
References
1.MolecularBiologyExperimentGuide,JinyuanLiu,etal.TsinghuaPress,2009
2.AddgeneVectorDatabase(http:
//www.lablife.org/vectordb)
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