Plasmid DNA.ppt

8/18/2019 Plasmid DNA.ppt

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DNA Ekstrakromosomal

(Plasmid DNA)

Soraya RahmanisaLab Biologi Molekuler FK Unila

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DNA ekstrakromosomal : DNA lain yangterdapat dalam sel di luar nukleus, yaitu :- DNA mitokondria,

- DNA kloroplas,

- DNA plasmid : merupakan molekul DNA tambahanatau elemen DNA ekstrakromosomal


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E. coli

Chromosome

Plasmids

Plasmids

• DNA molecules separate from chromosomal DNA

• Self-replicating

Electron micrograph of DNA

from a lysed E. coli cell

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Plasmid vs Episom

Episom merupakan unsur-unsur genetik bebas yangtelah dapat berkembang dalam sel bakteri baik

dalam keadaan autonom (menggandakan diri dandipindahkan tanpa bergantung kepada kromosom

bakteri) maupun pada keadaan terintegrasi (melekatpada kromosom bakteri, berperan serta bersamanya

dalam rekombinasi genetika dan dipindahkan bersama kromosom bakteri tersebut.

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• -plasmids! acilitate "acterial con#ugation

• $-plasmids! Confer resistance to anti"iotics or other to%ins

• Col-plasmids! Encode for colicines (potentially to%ic to other "acteria)• Degradati&e plasmids! Ena"le the "reakdo'n of certain su"stances• irulence plasmids! Causes the "acteria to act as a pathogen

Plasmid unctional Categories

Bacteria carrying a plasmid with the geneneomycin phosphotransferase are capableof surviving in the presence of the antibiotickanamycin

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http://en.wikipedia.org/wiki/Image:BacterConjugation.pnghttp://en.wikipedia.org/wiki/Image:BacterConjugation.pnghttp://en.wikipedia.org/wiki/Image:BacterConjugation.png


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!lasmid tipe 1" plasmid dengan #umlah kopian berganda denganpembagian acak$


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!lasmid tipe 2" plasmid dengan #umlahkopian sedikit dengan pembagian terarah$


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olecular *iology Applications for Plasmids+, Cloning of DNA fragments

++, Protein Production

+P./.0

promoter /ene of +nterest

Protein +nduction Plasmid

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Comparison of plasmid copy num"ers in E. coli

1igh copy

DNA yield

2ikelihood of mutation

Difficulty in cloning to%ic genes

2o' copy

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/al3 DNA-"indingDomain

*aitProtein

/al3 Acti&ationDomain

*ait ector Prey ector

2i"raryProtein

olecular *iology Applications for Plasmids+++, 4east .'o-1y"rid Assay!

.ests for protein-protein interactions

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olecular *iology Applications for Plasmids+, Agro"acterium-mediated Plant .ransformation

A means of performing plant genetic engineering

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Harvest cells by centrifugation

(pin )"000 rcf

Supernatant (clear)

Pelleted cellsE. coli culture(cloudy)

Discard supernatant *esidual media may interfere with downstream steps

Resuspend cells in buffer +horoughly resuspend cells" making sure that noclumps remain$ !1 buffer contains,

- +ris.l buffering agent - + metal chelator - *5ase degrades *5

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- issolves membranes

- Binds to and denatures proteins

5, Na61- enatures 5

*ecause plasmids are supercoiled7"oth DNA strands remain entangledafter denaturation

Lyse cells with SDS/NaOH solution

dding buffer !2 causes solution to become viscous

8, Sodium dodecyl sulfate

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Neutralie NaOH with potassiu! acetate solution

i%ing 'ith "uffer N9 causes a fluffy 'hite precipitate toform,

8, Potassium acetate : acetic acid solution - 5eutralies 5a78 renatures plasmid 5 - onverts soluble (( to insoluble !(

sodium dodecyl sulfate (SDS) potassium dodecyl sulfate (PDS) (H2O sol. = 10%) (H2O sol. < 0.02%)

5, /uanidine hydrochloride (/uCl) - haotropic salt9 facilitates 5 binding to silica in

later steps

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Separate plas!id DN" fro! conta!inants bycentrifugation

Supernatant contains! - Plasmid DNA

- Solu"le cellular constituents

Pellet contains! - PDS - 2ipids - Proteins

- Chromosomal DNA

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"dd cleared lysate to colu!n and centrifuge

.he high ionic strength and presence of chaotropic saltcauses DNA to "ind to the silica mem"rane7 'hile othercontaminants pass through the column

(ilica.gel membrane

entrifuge

:low through

discard

5ucleic acids

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#ash the silica !e!brane to re!ove residualconta!inants

*uffer P* contains isopropanol and /uCl

*uffer PE contains ethanol and .ris-Cl

entrifuge

!B ; contaminants

5ucleic acids

5ucleic acids

!B buffer

entrifuge

! ; contaminants

including residual


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Elute purified DN" fro! the colu!n

E* is 8; m .ris-Cl (p1


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anual alkaline lysis preparation of plasmid DNA

• 6rganic e%traction (optional)

=i> thoroughly with

an e?ual volume of

organic solvent

e.g. phenol" chloroform"

or phenol,chloroform

entrifuge

7rganic

?ueous

• Precipitate DNA 'ith isopropanol (8!8 &olume)(upernatant

!ellet

entrifuge0@ isopropanol;

precipitated 5

• >ash pellet 'ith 0;? Et61 (to remo&e salts)

• Dissol&e pellet 'ith .E (or other a@ueous solution)

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Assessing your plasmid preparation

8, uantify a"undance (A5B;) and purity (A5B; :A5


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Documents

Plasmid DNA.ppt