Agarose Gel Electrophoresis

Introduction

A technique used in Biochemistry and molecular biology to separate DNA or RNA molecules

Electrical current is used to create electric field across the agarose matrix

The negatively charged nucleic acid molecules will migrate across the gel matrix towards the positive electrode

Migration is based on their molecular size

Shorter molecules move faster and migrate further compared to the longer molecules because they can move through pores of the gel easily

Agarose gel electrophoresis will be then stained with ethidium bromide for DNA visualization

Discrete bands will be observed under the UV light

Agarose is preferred over acrylamide because it is less toxic and easy to handle although acrylamide gives better resolution

Agaropse Gel electrophoresis needs Buffer solution (TBA/ TAE), Agarose, power supply, UV lamp to visualize DNA in the gel, Molecular weight dye such as bromophenol blue, An ultraviolet-fluorescent dye such as Ethidium bromide

Components of Agarose Gel Electophoresis Buffer solution Agarose Ethidium bromide (5.25 mg/ml in H2O) Nitrile rubber gloves Bromophenol blue Glycerol Gel rack Comb UV lightbox

Recipe for Agarose

1. Add 0.5g of agarose into 50mL of TBE Buffer.

2. Microwave to dissolve agarose.3. Cool the agarose to 60°C.4. Pour mixture into tank.5. Place comb.

Principle

Agarose gel electrophoresis is a widely used method that separates molecules based upon charge, size and shape.

Useful in separating charged biomolecules such as DNA, RNA and proteins.

Proteins and nucleic acids are electrophoresed within an agarose "gel"

 The gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value.

A direct current power supply is connected to the electrophoresis apparatus and current is applied.

When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge.

~Molecules having a net negative charge migrate towards the positive electrode (anode) ~while net positively charged molecules migrate towards the negative electrode (cathode). ~In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode.

Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques.

Inject DNA ladder (molecular weight markers) into first well

Inject DNA samples into the following wells

DNA ladder and DNA samples are stained with loading dye (beta-

mercaptoethanol)

Apply current

Small DNA strands move faster and further through the gel

Stain with Ethidium bromide (EtBr)

Visible under ultraviolet (UV) light

Visualization & Result Analysis Staining

Ethidium Bromide, visualized under UV light

Blue excitable stain (eg. SYBR Green), visualized using blue light excitation source Safer Passes through transparent plastic and

glass

A 1% agarose 'slab' gel prior to UV illumination, behind a perspex UV shield. Only the marker dyes can be seen.

The gel with UV illumination, the ethidium bromide stained DNA glows orange.

Gel stained SYBR® Green I nucleic acid gel stain.

Digital photo of the gel after visualized under UV illumination.

Applications The preparation of the agarose gel is a

vital step in determining the well separated DNA bands. -It able to resolve different sizes of DNA fragments.

High concentration (1.5% or 2%) of agarose- resolves small DNAs (0.2-1.2kb)

Low concentration (0.7%) of agarose

- resolves large DNAs (8-10kb)

Applications Molecular biology based work:

- to purified the specific sizes of the DNA which are from the restriction enzyme digestion.- to obtain the cut plasmids (separating cut vector frm uncut one)

Prior to Southern blot transfer - to separate the genomic DNA frm the RE

digested DNA

Analysis the PCR product-to target the amplified DNA

Estimation of the size of the

studied DNA molecule

- by using the DNA ladder

(marker) of various known

band sizes

Determination of the DNA quantity & DNA quality:

(i) DNA quantity as identified by the λ DNA ladder with various known DNA in different bands (ruler)

(ii) DNA quality is directly can be observed frm the gel surface- any absence of streaking or fragment

(contaminated DNA molecule)

Other applications:

DNA fingerprinting (forensic science)- to target suspected DNA molecule from criminals

Alkaline agarose gel electrophoresis

- buffer used is substituted by NaOH

- to analyze ssDNA specifically

Pulse d field electrophoresis- current flow is periodically altered- to separate DNA ranging frm 50,000

to 5 million bp