Plasmid DNA Isolation

Experiment Goals

• Extraction of plasmid DNA from E. Coli.

• Analyze plasmid DNA by agarose gel electrophoresis and spectrophotometer.

Introduction• Many types of bacteria contain plasmid DNA.• Plasmids are extrachromosomal, double-stranded

circular DNA molecules separate from the chromosomal DNA.

• Certain plasmids replicate independently of the chromosomal DNA and can be present in hundreds of copies per cell.

• Generally containing1,000 to 100,000 base pairs. • Even the largest plasmids are considerably smaller

than the chromosomal DNA of the bacterium, which can contain several million base pairs.

Classification of plasmids by function

There are five main classes• Fertility-F-plasmids, Facilitate bacterial conjugation• Resistance-R-plasmids, which contain genes that can

build a resistance against antibiotics or poisons. • Col-plasmids, which contain genes that code for

bacteriocins, proteins that can kill other bacteria. • Degradative plasmids, which enable the digestion of

unusual substances, e.g., salicylic acid. • Virulence plasmids, which turn the bacterium into a

pathogen.

Plasmid Applications

• The plasmids used in transformation typically have three important elements:• A cloning site (a place to insert foreign

DNAs)• An origin of replication• A selectable marker gene (e.g. resistance to

ampicillin)

Plasmid Applications

Plasmid DNA isolation

• Isolation of plasmid DNA from bacterial cells is an essential step for many molecular biology procedures.

• Many protocols for large- and small-scale isolation of plasmids have been published.

• The plasmid purification procedures, unlike the procedures for purification of genomic DNA, should involve removal not only of protein, but also another major impurity: bacterial chromosomal DNA.

• Pick a single colony and inoculate in 5 ml of LB (Luria-Bertani) containing 20 mg/l ampicilin

• Incubate overnight at 37oC

• Centrifuge 1.5 ml of broth containing cells in a tube

• Discard supernatant

Overnight Culture Suspension

Plasmid DNA isolation

1. Inactivation of Bacteria

2. Lysis of cells/ denaturation of DNA

3. Precipitation of DNA

4. Separate plasmid DNA from contaminants

5. Precipitation of Plasmid DNA

6. Precipitation of proteins

7. Precipitate Plasmid DNA

1- Inactivation of Bacteria

• Resuspend cell pellet in 100 µl of GTE buffer (50mM Glucose, 25 mM Tris-Cl & 10mM EDTA, pH 8)• Glucose is added to increase the osmotic pressure

outside the cells• Tris is a buffering agent • EDTA protects the DNA from degradative

enzymes

• Vortex gently if necessary

• Dissolves membranes

• Binds to and denatures proteins

2. NaOH• NaOH rupture the cell and also denatures the DNA into single strands

2- Lysis of cells/ denaturation of DNA Add 200 µl of NaOH/ SDS lysis solution, invert tube 6-8 times 1. Sodium dodecyl sulfate

• Immediately add 150 µl of 5 M potassium acetate solution (pH 4.8)

1. Potassium acetate / acetic acid solution

• Neutralizes NaOH (renature plasmid DNA)

• Converts soluble SDS to insoluble PDS

sodium dodecyl sulfate (SDS) potassium dodecyl sulfate (PDS)

• Precipitate the genomic DNA

• Centrifuge for 1 minute at high speed

3- Precipitation of DNA

Separate plasmid DNA from contaminants by centrifugation

• Supernatant contains: - Plasmid DNA - Some cellular constituents • Sediment contains: - PDS - Lipids - Proteins - Chromosomal DNA

4- Separate plasmid DNA from contaminants

5- Precipitation of Plasmid DNA

• Transfer supernatant layer to a clean tube and add 0.5 ml of isopropanol on ice for 10 minutes

• Centrifuge at top speed for 1 minute

Add 0.5 ml of isopropanol to supernatant

Centrifuge

Supernatant

Incubate for 10 min. on ice Pellet

• Remove supernatant, dissolve pellet in 0.4 ml TE buffer

• Add 10 µl of RNAse solution, vortex & incubate at 37oC for 20 – 30 min.

6- Precipitation of proteins

• Add 300 µl of phenol/ chloroform/ Isoamyl alcohol • Vortex vigorously for 30 seconds • Centrifuge at full speed for 5 minutes

Mix thoroughly with an equal volume of organic solvent

phenol, chloroform,

Centrifuge

Organic

Aqueous

7- Precipitate Plasmid DNA

• Remove supernatant to a clean tube • Add 100 µl of 7.5 M ammonium acetate & 1 ml of absolute

ethanol to precipitate the plasmid DNA, incubate on ice• Mix and then centrifuge at full speed for 5 minutes

Supernatant

Pellet

CentrifugeAbsolute ethanol & ammonium acetate

precipitated DNA

• Wash pellet with 75% Ethanol (to remove salts), & dry pellet

• Dissolve pellet with TE (or other aqueous solution)

Quantifying Plasmid DNA

• Quantify DNA using UV absorbance

• DNA UV absorbance peaks at 260 nm

• protein UV absorbance peaks at 280 nm

• The ratio of the absorbance at 260 nm/280 nm is a measure of the purity of a DNA sample from protein contamination; it should be between 1.7 and 2.0

• The ratio of the absorbance at 260 nm/230 nm is a measure of the purity of a DNA sample from organics and/or salts; it should be about 2.0. Low 260/230 ratio indicates contamination by organics and/or salts

• http://www.dnatube.com/video/994/Plasmid-Isolation

• http://www.dnalc.org/resources/animations/transformation1.html

• http://www.learnerstv.com/animation/animation.php?ani=167&cat=biology

• http://resources.jorum.ac.uk/xmlui/bitstream/handle/123456789/13704/page83.htm?sequence=86

• http://www.sumanasinc.com/webcontent/animations/content/dna_library.swf

• http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html