2. dna preparation – mini prep

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