Gel Electrophoresis • based on motion of charged molecules in an electric field toward the

opposite charge. • Agarose gels (for larger fragments of DNA) or polyacrylamide

gels (for smaller fragments DNA or proteins) commonly used. Both made of a crosslinked matrix forming pores.

• At neutral pH each nucleotide has negative charge due to phosphate group.

• DNA runs to positive electrode• Length and Shape of the molecule influences how the DNA runs• Short pieces run faster• Big pieces run slowly• Conformation of plasmids clearly influences running speed• Slowest Relaxed circle, linear, supercoiled fastest

Agarose gel electrophoresis Agarose gel electrophoresis is an easy and common way of separating and analyzing DNA.Purpose of gel1. visualise,2. to quantify 3. isolate a particular band.

What percentage gel? Most agarose gels are made between 0.7% and 2%. 0.7% gel will show good separation (resolution) of large DNA fragments (5–10kb) 2% gel will show good resolution for small fragments (0.2–1kb). 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is better

Loading a gel and running a gel Steps1. The agarose gel with three wells

(S). 2. Loading DNA ladder (molecular

weight markers) into the first well. 3. Loading of samples into the second

and third well. 4. A current is applied. 5. The DNA moves toward the

positive electrode due to the negative charges on its phosphate backbone.

6. The DNA is not normally visible during this process, so the marker dye is added to the DNA to avoid the DNA being run entirely off the gel.

7. The marker dye has a low molecular weight, and migrates faster than the DNA, so as long as the marker has not run past the end of the gel, the DNA will still be in the gel.

The gel with UV illumination, the ethidium bromide stained DNA

glows pink An Agarose 'slab' gel

prior to UV illumination

Digital photo of the gel. Lane 1. Commercial DNA Markers (1kbplus), Lane 2. empty, Lane 3. DNA frag. just over 500 bases, Lane 4. smaller DNA frag.

How much DNA should I load?

• You want to be able to see the DNA bands under UV light in an ethidium-bromide-stained gel.

• A band is easily visible if it contains about 20ng of DNA.

E.g. you are digesting a plasmid that comprises 3kb of vector and 2kb of insert. You are using EcoRI (a common restriction enzyme) and you expect to see three bands: the linearised vector (3kb), the 5' end of the insert (0.5kb) and the 3' end of the insert (1.5kb

• To see the smallest band (0.5kb) you want it to contain at least 20ng of DNA.

• The smallest band is 1/10th the size of the uncut plasmid. • you need to cut 10x20ng, that is 200ng of DNA (0.2µg• your three bands will contain 120ng, 20ng and 60ng of DNA

respectively. • All three bands will be clearly visible on the gel and the biggest band

will be six times brighter than the smallest band. • If you cut the same plasmid with BamHI and BamHI only cuts the

plasmid once.• If you digest 200ng of DNA in this case then the band will contain

200ng of DNA and will be very bright and will be overloaded.

Loading buffer• Glycerol or dextran, buffer, plus colour• Weighs sample down and allows visualisation of loading.• Also allow visualization of how gel is running• Bromophenol blue migrates at a rate equivalent to 200–

400bp DNA. • Xylene cyanol migrates at approximately 4kb

equivalence. • Orange dye migrates at about 50bp DNA

What voltage?? • 10 volts per cm of gel length• If use too much current gel resistance, heating and gel

melts!

Visualization of DNA in gel• Stain DNA with Et Br• It intercalates between the bases• Fluoresces under UV light• Can also use Methylene blue to stain DNA but not as

sensitive