1. AGAROSE GEL ELECTROPHORESIS

2. Terms
Gel electrophoresis is a method that uses an electrical current and a gel matrix to separate molecules like DNA and proteins.
Buffer a solution containing either a weak acid and its salt or a weak base and its salt, which is resistant to changes in pH.

3. Agarose Gel Electrophoresis
This is a procedure that separates molecules on the basis of their rate of movement through agarose gel under the influence of an electrical field.
Separates DNA or RNA by:
size and/or
charge and/or
shape

4. Reagents and Supplies Weighing scale Spatula Flask Graduated cylinder
Microwave
Agarose
Buffer
Gel tray(s) and comb(s)
Gel box(es)
Power supply
DNA Staining solution
Photo doc. system

5. Electrophoresis Equipment
Power supply
Cover
Gel tank
Electrical leads 
Casting tray
Gel combs

6. Agarose Agarose is a linear polymer extracted from seaweed.
An agarose gel is used to slow the movement of DNA .
Within an agarose gel, linear DNA migrate inversely proportional to their molecular weight.

7. Resolution of linear DNA fragments in agarose gel
% Agarose (w/v) Size Range (kb)
for Optimal Separation

8. Buffer Systems
Weak acids and/or bases that do not dissociate completely.
Purposes of buffer:
Maintain pH.
Generate ions consistently to maintain current & keep resistance low.
TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA
TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA
TBE resolves low MW fragments better than TAE.
TAE resolves high MW fragments better than TBE
Tris (T) is a weak base.
Acetic (A) acid & boric (B) acid are weak acids.

9. Pouring a horizontal agarose gel

10. Visualization Monitoring the progress of the electrophoresis
Tracking dyes are visible to naked eye during run
Xylene cyanol (migrates with ~5.0 kb fragments)
Bromophenol blue (migrates with 300 bp fragments)
Orange G (migrates with fragments of ~50 bp)
But
Mobility of tracking dyes can vary substantially depending on agarose
Concentration
Type

11. Ethidium bromide staining
Binds to ds DNA by intercalation between stacked bases.
Used to visualize DNA with UV light.
***CAUTION!
UV light damages eyes and skin! Wear goggles and/or face shield.
Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

12. + - How fast will the DNA migrate? Strength of the electrical field
Size of the DNA
Buffer
Concentration of agarose gel used
DNA + -
Power
small
large

13. What factors affect mobility of linear ds DNA?
Pore size of the gel
 [agarose]   pore size
 pore size   friction   mobility
Voltage across the gel
 voltage   mobility
Length of the DNA molecule
smaller molecules generate less friction and so move faster

14. Factors affecting resolution
Resolution = separation of fragments
The “higher” the resolution, the more space between fragments of similar, but different, lengths.
Resolution is affected by
agarose type & concentration
salt concentration of buffer or sample
amount of DNA loaded in the sample
voltage

15. Why run an agarose gel? Determine the quality or quantity of DNA
Estimate the size of DNA molecules
Purification of DNA
Analyze PCR products
Molecular diagnosis or genotyping

16. Genomic DNA
M = 1kb + DNA ruler

17. PCR products
M 3a 4a 4b 1a 1b 2a

18. Msel digestion of PCR products

19. PCR products

20. PCR products

21. Genomic DNA

22. Trouble shooting Smearing torn sample wells
voltage too high for large fragments
too much DNA
Gel melts
voltage too high
ionic strength too low
Poor resolution
wrong agarose concentration
small bands are fuzzy –diffusion of the DNA and broadening of the band