Gel Electrophoresis

Agar gel electrophoresis of Nucleic acids

1. Electrophoresis buffer 2. Loading buffer 3. Ethidium bromide (staining) 4. Transilluminator (UV light box) 5. Submarine gel

How does GE work?

1. Molecules are forced across a span of gel 2. Electrodes at either end of the gel provide the driving force 3. Size of a molecule determines how rapidly it moves through a gelatinous medium. 4. The charged particles migrate either to the cathode or the anode. 5. The electric field repels the molecules from one electrode while the other electrode simultaneously attracts the molecules. 6. The pore size of the gel acts as a "molecular sieve," separating the molecules by size.

Daltons to base pairs

650 daltons=1 bp

Ethidium bromide

A fluorescent dye used for staining nucleic acids. EB intercalates between base pairing of nucleic acids and allows very convenient detection of DNA fragments in gels.

What is gel electrophoresis?

A method that separates macromolecules - either nucleic acids or proteins- on the basis of size

What is agarose?

A natural colloid extract from seaweed. Very fragile and easily destroyed by handling. Have very large pore size and are used primarily to separate very large molecules with a molecular mass greater than 200 kdal (kilo-daltons)

Agarose compared to polyacrylamide.

Agar can be processed faster, but their resolution is inferior. That is, the bands formed in the agar gels are fuzzy and spread far apart. This is a result of pore size and it cannot be controlled.

How to pour a gel

Agar is heated until about 60 deg C and poured into casting tray containing a comb. Gel is then allowed to cool until solidified.

What are the factors that effect electrophoresis?

Agarose Concentration Voltage Electrophoresis buffer Effects of Ethidium Bromide

What are the 2 types of gels used?

Agarose and Polyacrylamide

How are biological molecules able to move based on charge?

Amino acids, peptides, proteins, nucleotides, and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charges species either as cations (+) or anions (-).

Voltage (how does it effect electrophoresis?)

As the voltage applied to a gel is increased, DNA fragments migrate faster. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel. (the cm value is the distance between the 2 electrodes, not the length of the gel)

Reptation

Being forced through and finding their way through the bigger pores (like a snake).

What concentrations are agarose gel at?

Between 1% and 3%

What concentrations are polyacrylamide gels at?

Between 3% to 30%. Precise pore sizes can be obtained usually from 5 to 2,000 kdal. Ideal range for gene sequencing, protein,polypeptide, and enzyme analysis.

What are 2 types of tracking dyes

Bromophenol blue (300bp) and xylene cyanol (4000 bp) dyes migrate through agarose gels at roughly the same rate as double stranded DNA fragments of the respective bp.

What are the charges of anode and cathode?

Cathode: Negative Anode: Positive

After gel has solidified

Comb is removed carefully not to disturb wells. Samples containing loading buffer are pipetted into wells.

Loading buffer

Contains something dense (ex: glycerol) to allow the sample to "fall" into the sample wells. One or two tracking dyes, which migrate in the gel and allow monitoring how far the electrophoresis has proceeded.

Electrophoresis buffer and its effect on electrophoresis

DNA fragments will migrate at somewhat different rates in the 2 buffers (TAE and TBE) due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity.

Agarose concentration (how does it effect electrophoresis?)

Different concentrations of agarose, different sizes of DNA fragments. High concentrations of agar facilitate separation of small DNAs, while low concentration of agar allow resolution of larger DNAs.

What happens if you use concentrated buffer (like a 10x stock solution)

Enough heat may be generated in the gel to melt it.

How do polyacrylamide gradient gels perform?

Gradient gels provide continuous decrease in pore size from the top to the bottom of the gel, resulting in thin bands. B/c of this banding effect, detailed genetic and molecular analysis can be performed on gradient gels.

Proteins vs. Nucleic Acids

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.

Effects of ethidium bromide on electrophoresis

It can be incorporated into agar gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. Binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.

Migration of DNA fragments (linear)

Migrate through agar gel with a mobility inversely proportional to the log (MW). If you plot the distance that DNA fragments have migrated against the log (MW), a straight line will appear.

Migration of DNA fragments (circular)

Migrates differently form linear DNAs of the same mass. Uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Most preparation of uncut plasmid contain at least 2 topologically-different forms of DNA, correspondng to supercoiled forms.

What is the charge of ssDNA?

Negative due to phosphate groups. The charge to mass ratio is constant.

Running of a gel

Once samples are loaded, desired voltage is applied, nucleic acids runs towards the red anode, you can confirm that current is flowing by observing bubbles coming off the electrodes. The distance DNA has migrated in gel can be judged by visually monitoring migration of the tracking dyes. After run, gel can be viewed with transilluminator for observation of nucleic acid bands.

What if macromolecules are the same size?

Separated based on charge ratio, substances will not separate and will move together in an aqueous environment. Thus we use a gel matrix.

SDS-PAGE

Sodium dodecyl sufate polyacrylamide gel electrophoresis Special form of PAGE that employs a detergent to denature the protein. Proteins assume a rod like shape in the presence of SDS. The net result is that the proteins have similar shapes and charge-to-mass ratios and are therefore separated by gel filtration effects. They now separate just according to size.

How do peptides/proteins have an overall negative charge?

Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by "wrapping around" the polypeptide bone.

How are agarose gels formed?

Suspending dry agarose in aqueous buffer, then boiling the mixture until a clear solution forms. This is poured and allowed to cool to room temperature to form a 11 rigid gel.

What is electrophoresis?

The migration of charged particles under the influence of an electric field

Polyacrylamide gel electrophoresis (PAGE)

The molecular separation is based on sieving effects as well as electrophoretic mobility.

What happens if you use water instead of buffer?

There will be essentially no migration of DNA in the gel.

What are the common electrophoresis buffers?

Tis-acetate-EDTA (TAE) Tris-borate-EDTA (TBE)*

What is polyacrylamide?

Used for skin electrodes and in soft contact lenses. Pore size may be varied to produce different molecular sieving effects for separating proteins of different sizes. **Gels can be cast in a single percentage or with varying gradients.** Greater flexibility and more sharply defined banding than agar gels.

Separation of large (macro) molecules depends upon what 2 forces?

charge and mass