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DNA Ekstrakromosomal
(Plasmid DNA)

Soraya Rahmanisa
Lab Biologi Molekuler FK Unila

05/03/19
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.

05/03/19
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 Bait Gal4 Activation Library

Domain Protein Domain Protein

Bait Vector Prey 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 Pelleted cells
(cloudy)

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) potassium dodecyl sulfate (PDS)

(H2O sol. = 10%) (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 Aqueous

an equal volume of
organic solvent Centrifuge
e.g. phenol, chloroform,
or phenol:chloroform
Organic

• Precipitate DNA with isopropanol (1:1 volume)

Supernatant

50% isopropanol
+
Centrifuge
precipitated DNA

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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