1ml of overnight cultureRemoved, spinned andsupernatant removed.Bacteria pellet resuspendet inbuffer that does not kill cells
Note: Alkaline liquid: mix of NaOH andSDSif DNAis too long insolution withhigh pH: Hydolysis à destroyed
Resin in columnIs positivley charged:Binds negative chargeof plasmid DNAbackbone
The lysate is neutralized bytheaddition ofacidicpotassium acetate; Thehighsalt concentration causesPotassium dodecyl sulfate toprecipitate, and thedenatured proteins,chromosomal DNA,andcellular debris becometrapped in salt–detergentcomplexes. Plasmid DNA,being smaller and covalentlyclosed, renatures correctlyand remains in solutionCentrifugation an high speed(ca. 13.000 rpm); cell debriesand genomic DNAprecipitate;small DNAmolecules (plasmidremain in supernatant)
2.DNAPREPARATION– Miniprep
TheuseofcolumnsResults invery pureplasmid DNA.“sequence grade”
Silica
3.CONTROLDIGESTTOINDETIFYSUCCESSFUL CLONINGEVENT
pBluescript(2900bp)
pBluescript+2400ntinsert
+EcoRI +EcoRI
2900bp (linearized) 2900bp2400bp
pBluescript
pBluescript+
Insert
2900bp
2400bp
+alkaline phosphatase
Ligating 2fragements withDNALigase
2900nt(linearizzato)2400nt
Attention:All involvedoverhangs arecompatibleà insert canbe“ligated”into vector inboth orientations
-OH
HO-
+alkaline phosphatase
2900nt2400nt
EcoRI
Note:5’overhangsofinsert andlinearized plasmidsarecompatible;both havebeen cut withEcoRI.Ligase covalently linksbothmoleculesEcoRI sites arereconstiuted andnowflank theinsertsequence!!!
PstI
3.DNAPREPARATIONANDCONTROLDIGEST– ORIENTATIONOFINSERT?
P--P
3.DNAPREPARATIONANDCONTROLDIGEST– ORIENTATIONOFINSERT?
3possible products ofligation
Blue-white screeningusing forexample thepBluescript vector
Whitecolony Bluecolony Whitecolony
HOWCANWEDETERMINETHE
ORIENTATIONANDIDENTITYOFTHE
INSERT?
Insert DNAsequence is knownà Iknow all restriction sites
Blue-white selection is OK…....butdoestheplasmidreallyhasthecorrectinsert???
5’
5’
5’
5’3’ 3’
3’
PstI
Insert2400nt
300 2100
EcoRI EcoRI EcoRI EcoRIPstIPstI PstI PstIPstI
nucleotideposition300
3.DNAPREPARATIONANDCONTROLDIGEST– ORIENTATIONOFINSERT?
ACONTROLDIGESTISPERFORMEDONMULTIPLECOLONIESOBAINEDFROMCLONINGEXPERIMENT(5-10)
Ingeneral:pick 6-10white colonies withsterilepipettetip
Next day:harvest bacteria bycentrifugationandprepare plasmid DNA
6-10minipreps
Insert2400nt
300 2100
EcoRI EcoRI EcoRI EcoRIPstIPstI PstI PstIPstI
nucleotideposition300
Length
marker
undigested
Colony
1/EcoR
I
Colony
1/PstI
Colony
2/PstI
Colony
3/PstI
Colony
4/PstI
Colony
5/PstI
Colony
6/PstI
300nt
5000nt
2100nt
3200nt
Insert:2400ntPlasmid:2900nt
Cut withrestrictionenzymethat result asymetricdigestionproducts
OptionA. OptionB.
A A AB BB
3.DNAPREPARATIONANDCONTROLDIGEST– ORIENTATIONOFINSERT?
EcoRIEcoRI
EcoRILigase
SmaISmaI
EcoRVLigase
EcoRI
BamHI/EcoRILigase
BamHIEcoRI
PstI/EcoRILigase
BamHI
OVERVIEWOVERONCLONINGSTRATEGIES
1 2
3
BamHI EcoRI BamHI/EcoRILigase
4
PCR
DNACLONINGWITH2COHESIVEOVERHANGS
EcoRI:
BamHI: G/GATCCCCTAG/G
G/AATTCCTTAA/G
G A T C C
G
C C C T A G
G
cut withBamHI andEcoRI
cut withBamHI andEcoRI
+19ntfragment=vetor sequence fromBamHI toEcoRI site(eliminated during gelrun/purification
DIRECTIONALCLONINGà Alwayspreferredcloning strategy
P--P
-HO-P
nobasepairing
-HO
EcoRI:
BamHI: G/GATCCCCTAG/G
G/AATTCCTTAA/G
G A T C C
G
C C C T A G
G
cut withBamHI andEcoRI
cut withBamHI andEcoRI
+19ntfragment=vetor sequence fromBamHI toEcoRI site(eliminated during gelrun/purification
DNACLONINGWITH2COHESIVEOVERHANGS
1. EcoRI/BamHI digest toobtain insert2. EcoRI/BamHI digest toobtain linearized pBluescript3. Gelrun andpurification ofrelevant DNAfragments4. Setupligation (plasmid:insert =1:3)5. Transformcompetent bacteria;plate onagarplates +X-GAL,IPTG,ampicillin à pick white colonyàmake liquid bacterial culture6. Plasmid preparation andcontroldigest toverify presence ofcorrect insert7. IMPORTANT:NOALKALINEPHOSPHATASEREQUIREDà EcoRI andBamHI donot represent cohesive ends!!8. IMPORTANT:ORIENTATIONOFINSERTISALWAYSTHESAME!!!
P--P
-HO-P
-HO
EcoRIEcoRI
EcoRILigase
SmaISmaI
EcoRVLigase
EcoRI
BamHI/EcoRILigase
BamHIEcoRI
PstI/EcoRILigase
BamHI
OVERVIEWOVEROTHERCLONINGSTRATEGIES
1 2
3
BamHI EcoRI BamHI/EcoRILigase
4
PCR
SmaI:
EcoRV:
CCC/GGGGGG/CCC
GAT/ATCCTA/TAG
G A T C CG
G G GC C Ccut withSmaI
cut withEcoRV
+Linearized withEcoRVanddephosphorylationusing alkalinephosphatase
DNACLONINGWITHBLUNTENDS
1. SmaI digest toobtain insert2. EcoRV digest +alkaline phosphatasetreatmenttoobtain linearized pBluescript (that connot re-ligate)3. Gelrun andpurification ofrelevant DNAfragments4. Setupligation (plasmid:insert =1:3(5))5. Transformcompetent bacteria;plate onagarplates +ampicillin àpick colonyàmake liquid bacterial culture6. Plasmid preparation andcontroldigest toverify presence ofcorrect insert à insert canbeinserted inboth orientations!!7. IMPORTANT:SmaI sites arefused toEcoRV siteè cannot becleaved bySmaI orEcoRV8. Chose resitrction enzymes forcontroldigest that allow toidentify orientation ofinsert.
C C CG G G
GATGGGCTACCC
CCCATCGGGTAG
P--P-OH
OH-
EcoRIEcoRI
EcoRILigase
SmaISmaI
EcoRVLigase
EcoRI
BamHI/EcoRILigase
BamHIEcoRI
PstI/EcoRILigase
BamHI
OVERVIEWOVEROTHERCLONINGSTRATEGIES
1 2
3
BamHI EcoRI BamHI/EcoRILigase
4
PCR
DNACLONINGWITHMODIFICATIONOFOVERHANGS
BamHIEcoRI
PstI/EcoRILigase
(noBamHI site inMCS)
4
Lets assume:BamHI is notpresent inpBS
cut withPstI/EcoRI
CTCGA/GG/AGCTC
PstI:
EcoRI: G/AATTCCTTAA/G
BamHI: G/GATCCCCTAG/G
EcoRI: G/AATTCCTTAA/G
BamHI EcoRIG A T C C
G
NOTCOMPATIBLEàmake blunt
COMPATIBLE
DNACLONINGWITHMODIFICATIONOFOVERHANGS
àModification of5’overhangofBamHI siteà convert overhang toblunt endà Modifcation of3’overhangofPstI siteà convert overhang toblunt end
à è Blunt – Blunt ANDEcoRI – EcoRI ligation
INSERT
VECTOR 3’overhang
5’overhang
TheKlenow fragment is alargeprotein fragmentproduced when DNApolymerase IfromE.coliisenzymatically cleaved bytheprotease subtilisin.Firstreported in1970.It retains the5'→3'polymerase activity andthe3’→5’exonucleaseactivity forremoval ofprecodingnucleotides andproofreading,but loses its 5'→3'exonucleaseactivity.Theother smaller fragment formedwhen DNApolymerase IfromE.coliis cleaved bysubtilisin retains the5'→3'exonucleaseactivity butdoes not have theother twoactivities exhibited bytheKlenow fragment (i.e.5'→3'polymerase activity,and3'→5'exonucleaseactivity).
à Synthesis ofdouble-stranded DNAfromsingle-stranded templates
à Filling inreceded 3'ends ofDNAfragments tomake5'overhang blunt
à Digesting away protruding 3'overhangà Preparation ofradioactiveDNAprobes
DNACLONINGWITHMODIFICATIONOFOVERHANGS
TheKlenow fragment
DNAPolymerase I(E.Coli)- 5'→3'polymerase activity- 3’→5’exonucleaseactivity- 5'→3'exonucleaseactivity
5’ 3’5’3’
Klenow:inpresence ofdNTP:synthesisInabsence ofdNTP:3à’5’exonucleaseactivty
PolExo