DNA Isolation &

Agarose Electrophoresis

Lecture2

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Nucleic Acid purification can be performed starting from various biological materials:

• Blood

• Tissues

• Bacteria

• Fungi

• Animal cells

• Plant cells

• Dung/Stool

• Agarose gels

• Type of starting material

• Expected yield

• Sample age (I.e. quality)

• Time required for procedure

• Cost

• Quality control issues

Technique used depends on …

General Extraction Protocols Include:

1. Tissue Digestion / Cell Lysis2. Chelating and Proteinase Agents3. Nucleic Acid and Protein Separation4. Clean-up

Tissue Digestion / Cell Lysis

• Disrupt cells or tissue

• Avoid shearing forces that fragment DNA

• Method used depends on cell type

Examples of methods:

• enzyme-based lysozymes• sonication• high-salt hypotonic bursting• SDS detergent membrane disruption

QIAgen DNeasy DNA Kit1. Treat with Proteinase K

- digest proteins and inhibit nucleases

2. Lysis: QIAgen Buffer AL (guanidium-HCl) - hypertonic high salt solution

3. Precipitation of DNA with EtOH

4. Spin column - DNA binds to silica membrane

5. Wash

6. Elution 1 and 2, with warm Buffer AE (Tris-EDTA)

Agarose Gel Electrophoresis

Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field.

“Run to the Red”

DNA is negativelycharged because of thesugar-phosphate backbone.

DNA will be attracted to the positively charged pole (RED) and repelled from negatively charged pole (BLACK)

Agarose

Polymerizes to form a solid ‘gel’ which is porous allowing for the movement of DNA.

Small DNA fragments pass more easily through pores than large ones.

D-galactose 3,6-anhydroL-galactose

Scanning Electron Micrograph of Agarose Gel

An agarose gel is prepared by combining agarose powder and a buffer solution.

Agarose

TBE Buffer

Flask for boiling

Special thanks to Michael Clark (University of Rochester) for these images.

Casting trayGel combs

Power supply

Gel tank

Cover

Electrical leads

Electrophoresis Equipment

Seal the edges of the casting tray with masking tape and put in one comb

Preparing the Casting Tray

Agarose 1x TBE Buffer

Combine the agarose powder and 1x TBE buffer. Use a flask that is several times larger than the volume of buffer.

Melting the Agarose

Insoluble at room temp Soluble once boiled

Once the gel has reaches about 55 oC, Add 4-5 µl of Ethidium Bromide dye to pre-stain.

Ethidium Bromide

Warning! Mutagen!

Intercalates into nucleic acidand fluoresces under UV light

Carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.

Pouring the gel

The gel comb should be submerged in the melted agarose solution.

When cooled (> 20 mins), the agarose polymerizes, forming a flexible gel. Carefully remove the combs and tape.

Place the gel in the electrophoresis tank (aka gel box).

“Run to the Red”

Cathode(black -)

Anode(red +)

TBE buffer

Add enough 1x TBE buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.

6X Loading Buffer: Bromophenol Blue (colour) Glycerol (weight)

Sample Preparation

Mix the samples of DNA with the 6X sample loading buffer. This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells.

Loading the Gel

Carefully place the pipette tip inside a well and gently/slowly expel the sample. The sample should sink into the well.

Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. When the power is turned on, bubbles should form on the electrodes wires in the electrophoresis chamber.

Running the Gel

wells Bromophenol Blue

Cathode(-)

Anode(+)

After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.

DNA(-)

Visualizing the DNA under UV light

1 2 3 4

2.0

0.6

Eric’s Quote of the Week

‘Never throw away your samples until the end of the experiment.’

Dumpster diving SUCKS!

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