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DNA Ekstrakromosomal
(Plasmid DNA)

Soraya Rahmanisa
Lab Biologi Molekuler FK Unila

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DNA ekstrakromosomal : DNA lain yang

terdapat dalam sel di luar nukleus, yaitu :

- DNA mitokondria,
- DNA kloroplas,
- DNA plasmid : merupakan molekul DNA tambahan

atau elemen DNA ekstrakromosomal

Plasmids

E. coli
Chromosome

Electron micrograph of DNA

from a lysed E. coli cell

Plasmids
DNA molecules separate from chromosomal DNA
Self-replicating
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Plasmid vs Episom
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Episom merupakan unsur-unsur genetik bebas yang

telah dapat berkembang dalam sel bakteri baik

dalam keadaan autonom (menggandakan diri dan
dipindahkan tanpa bergantung kepada kromosom
bakteri) maupun pada keadaan terintegrasi (melekat
pada kromosom bakteri, berperan serta bersamanya
dalam rekombinasi genetika dan dipindahkan
bersama kromosom bakteri tersebut.

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Plasmid Functional Categories

F-plasmids: Facilitate bacterial conjugation

R-plasmids: Confer resistance to antibiotics or other toxins

Bacteria carrying a plasmid with the gene
neomycin phosphotransferase are capable
of surviving in the presence of the antibiotic
kanamycin
Col-plasmids: Encode for colicines (potentially toxic to other bacteria)
Degradative plasmids: Enable the breakdown of certain substances
Virulence plasmids: Causes the bacteria to act as a pathogen

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Plasmid tipe 1, plasmid dengan jumlah kopian berganda dengan

pembagian acak.

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Plasmid tipe 2, plasmid dengan jumlah

kopian sedikit dengan pembagian terarah.

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Molecular Biology Applications for Plasmids

I. Cloning of DNA fragments

II. Protein Production

+ IPTG
T7
promoter

Gene of Interest

Protein Induction Plasmid

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Comparison of plasmid copy numbers in E. coli

High copy

Low copy

DNA yield
Likelihood of mutation
Difficulty in cloning toxic genes
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Molecular Biology Applications for Plasmids

III. Yeast Two-Hybrid Assay:
Tests for protein-protein interactions
Gal4 DNA-binding
Domain

Bait
Protein

Gal4 Activation
Domain

Library
Protein

Prey Vector

Bait Vector

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Molecular Biology Applications for Plasmids

IV. Agrobacterium-mediated Plant Transformation
A means of performing plant genetic engineering

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How to purify plasmid DNA using

silica-based columns

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Harvest cells by centrifugation

Spin ~5,000 rcf

Supernatant (clear)
E. coli culture
(cloudy)

Pelleted cells

Discard supernatant

Residual media may interfere with downstream steps

Resuspend cells in buffer

Thoroughly resuspend cells, making sure that no

clumps remain. P1 buffer contains:
Tris-Cl (buffering agent)
EDTA (metal chelator)
RNase A (degrades RNA)

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Lyse cells with SDS/NaOH solution

Adding buffer P2 causes solution to become viscous
1. Sodium dodecyl sulfate
Dissolves membranes

Binds to and denatures proteins

2. NaOH
Denatures DNA
Because plasmids are supercoiled,
both DNA strands remain entangled
after denaturation

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Neutralize NaOH with potassium acetate solution

Mixing with buffer N3 causes a fluffy white precipitate to
form.
1. Potassium acetate / acetic acid solution
Neutralizes NaOH (renatures plasmid DNA)
Converts soluble SDS to insoluble PDS

sodium dodecyl sulfate (SDS)

(H2O sol. = 10%)

potassium dodecyl sulfate (PDS)

(H2O sol. < 0.02%)

2. Guanidine hydrochloride (GuCl)

Chaotropic salt; facilitates DNA binding to silica in
later steps

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Separate plasmid DNA from contaminants by

centrifugation
Supernatant contains:
- Plasmid DNA
- Soluble cellular constituents
Pellet contains:
- PDS
- Lipids
- Proteins
- Chromosomal DNA

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Add cleared lysate to column and centrifuge

Centrifuge
Nucleic acids
Silica-gel membrane

Flow through
(discard)

The high ionic strength and presence of chaotropic salt

causes DNA to bind to the silica membrane, while other
contaminants pass through the column

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Wash the silica membrane to remove residual

contaminants
Buffer PB contains isopropanol and GuCl

Centrifuge
PB buffer

Nucleic acids

Nucleic acids

PB + contaminants

Buffer PE contains ethanol and Tris-Cl

Centrifuge
PE buffer

Nucleic acids

Nucleic acids

PE + contaminants
(including residual GuCl)

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Elute purified DNA from the column

Buffer EB should be added directly
to the membrane for optimal DNA
recovery and to avoid possible
EtOH contamination (from residual
PE buffer)

EB is 10 mM Tris-Cl (pH 8.5). TE or dH2O may

also be used.
Centrifuge
EB buffer
Nucleic acids

EB + DNA

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Manual alkaline lysis preparation of plasmid DNA

Organic extraction (optional)

Mix thoroughly with
an equal volume of
organic solvent

Aqueous

Centrifuge

e.g. phenol, chloroform,

or phenol:chloroform
Organic

Precipitate DNA with isopropanol (1:1 volume)

Supernatant
50% isopropanol
+
precipitated DNA

Centrifuge
Pellet

Wash pellet with 70% EtOH (to remove salts)

Dissolve pellet with TE (or other aqueous solution)

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Assessing your plasmid preparation

1. Quantify abundance (A260) and purity (A260/A280)
2. Verify by restriction digestion
3. Run undigested plasmid to see if it is mostly
supercoiled

supercoiled
denatured

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