Electrophoresis. The purpose of electrophoresis is to separate molecules of DNA, RNA or protein. Separation can be based upon: Size Shape Isoelectric point (protein only). Separation is achieved by using a matrix (gel) and an electromotive force to propel the molecules through the gel .
1. Preparation of the gel tray
2. Melting the agarose in a buffer solution
4. Pouring the molten agarose into the casting tray with a comb
5. Letting the gel solidify
6. Submerging the solid gel in buffer solution in the gel box
7. Removing the comb to reveal the wells in the gel
8. Loading the samples into the wells
9. Applying the electric current and “running” the gel
10. Analyzing the samples separated in the gel
(note: these are estimates, the volumes will vary with the apparatus used!
Note: some electrophoresis apparatuses come with gel-casting boxes
This is commercially available
Has the consistency of fine sugar
Weighing out the powder
Use a weigh boat and scale
Tare the scale with the empty container
Add an appropriate amount of agarose
TAE and TBE buffers are available commercially
Remove and carefully stir by swishing the solution (the solution may be superheated so wear oven gloves!)
Return solution to the microwave and keep heating and swishing for 30 second intervals until all the agarose powder has dissolved
Note: this can also be done using a hot-plate!
Let the gel set until it become opaque
Carefully remove the comb.
Place the cover on the tray box and attach the electrodes
Remember “DNA runs to Red”
The electrode near the wells attaches to the negative terminal (cathode) and the other electrode attaches to the positive terminal (anode).
Once the wires are attached, turn on the voltage.
As a general rule: 5 Volts per cm (distance between electrodes on the tank)
Distance between electrodes