Gel Electrophoresis

What is Gel Electrophoresis?

Gel electrophoresis separatesmolecules on the basis of their charge and size. The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel acts as a sieve to to retard the passage of molecules according to their size and shape.

BIOTECHNOLOGY One of the basic tools of modern biotechnology

is gene splicing.

This is the process of removing a functional DNA fragment ( a gene) from one organism and combining it with the DNA of another organism to study how the gene works.

The desired result is to have the new organisms carry out the expression of the gene that has been inserted.

Restriction Enzymes

The ability to cut and paste DNA predictably is due to the use of restriction enzymes.

They were first identified in and isolated from the bacteria that use them as a natural defense mechanism to cut up the invading DNA of bacteriophages – viruses that infect bacteria.

They are named for the

Gel Electrophoresis

Electrophoresis is the movement of molecules by an electric current.

Nucleic acid moves from a negative to a positive pole.

Gel Electrophoresis

When DNA is applied to a macromolecular cage or gel such as agarose or polyacrylamide, its migration under the pull of the current is impeded.

Gel Electrophoresis

Slab gel electrophoresis can have either a horizontal or vertical format.

Sample is introduced into wells at the top of the gel.

Very Large DNA Molecules are Separated by Pulsed Field Gel Electrophoresis (PFGE).

The movement of molecules is impeded in the gel so that molecules will collect or form a band according to their speed of migration.

500 bp

200 bp

50 bp

% agarose: 2% 4% 5%

500 bp

200 bp

50 bp

500 bp

200 bp

50 bp

The concentration of gel/buffer will affect the resolution of fragments of different size ranges.

How does electrophoresis work?

• The gel is made from agar• DNA is a negative molecules• Molecules sort based on•Charge•Size•shape

What is agar?

Agar comes from sea weed.

What is it used for?

The gel is 1% agarous and has no electrical charge.

Electrophoresis Buffers

Carry current and protect samples during electrophoresis.

Tris Borate EDTA (TBE), Tris Acetate EDTA (TAE), Tris Phosphate EDTA (TPE) used most often for DNA.

10 mM sodium phosphate or MOPS buffer used for RNA.

Buffer additives modify sample molecules.– Formamide, urea (denaturing agents)

How does it work?

• DNA is cut into smaller fragments.

• Loading dye is used to indicate the fragments of DNA are behind the dye

• The negative DNA molecule is attracted to the positive electrode.

•The smallest fragments move the greatest distance.

Electrophoresis Equipment

Horizontal or submarine gel

Electrophoresis Equipment

Combs are used to put wells in the cast gel for sample loading.

– Regular comb: wells separated by an “ear” of gel

– Houndstooth comb: wells immediately adjacent

Running a Gel

Use the proper gel concentration for sample size range.– 0.5–5% agarose– 3.5–20% polyacrylamide

Use the proper comb (well) and gel size.

Running a Gel

Load sample mixed with tracking dye (dye + density agent).

Running a Gel

Detect bands by staining during or after electrophoresis

Ethidium bromide: for double-stranded DNA SyBr green or SyBr gold: for single- or

double-stranded DNA or for RNA Silver stain: more sensitive for single- or

double-stranded DNA or for RNA and proteins

Procedure

Remove comb and observe wells. Place carbon paper in each end of the tray. Cover with buffer, making sure the allow buffer to

overflow into each end of the tray. Load gels. Connect the electrodes. Turn on power supply. Allow gels to run – make sure you see bubbles

coming from the electrodes.

PROCEDURE (CONTINUED)

It will take about 30 minutes for the gel to run.

Turn off power supply and remove electrodes.

Pour off buffer into the designated container.

Carefully remove gel from gel box and place in glad container and cover with stain.

Store in appropriate location.

What is significant about the bubbles?

They indicate that electrolysis of water is taking place.

One electrode will have a lot of bubbles and the other will have a lesser amount. Why the difference?

The formula for water is H2O and the splitting of the molecule will produce twice as many atoms of hydrogen.

Summary

Electrophoresis is used to separate molecules by size and/or charge.

Nucleic acid fragments can be resolved on agarose of polyacrylamide gels.

PFGE is used to resolve very large DNA fragments. CGE is more rapid and automated than slab gel

electrophoresis. The choice of electrophoresis method depends on

the type and size of sample.