Team:Washington/AGAROSE GEL

From 2013.igem.org

Revision as of 03:41, 21 September 2013

""""

Agarose Gel Electrophoresis

General Procedure
1.    Cast a gel
2.    Place it in gel box in running buffer
3.    Load samples
4.    Run the gel
5.    Image the gel

Casting Gels
The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide:

1. Measure out the appropriate mass of agarose into a beaker with the appropriate volume of TAE buffer (see the documentation for your gelbox -- 50mL makes a good, thick gel for a 7x10cm gelbox). We are currently using a ~35mL box.
2. Microwave until the agarose is fully melted. This depends strongly on your microwave, but 90 seconds at full power or 3 minutes at half power seems to provide decent results. As long as you do not burn the agarose and nothing bubbles over, this step is robust.
3. From here on, a heat protective glove should be used any time the heated flask must be touched!
4. Let the agarose cool on the bench for ~5 minutes, to not warp tray and prevent leaks.
5. At this point add your DNA visualization reagent, e.g., ethidium bromide SYBR Safe at a volume appropriate for the amount of melted agarose (3uls for 30mLs). This amount will depend on the concentration of the stock solution of the stain (10,000X).
6. The flask will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles You should therefore periodically swirl the flask while the agarose is cooling.
7. Pour the agarose solution into the gel casting apparatus. A pipette tip should be used to pop or shove to the side any bubbles. Remove bubbles if they obscure the gel bands.
   
8. After bubbles have been popped, the comb should be inserted, and the gel should be allowed to cool for about 30 minutes, until solid.
9. Because removal of the comb too early can cause the wells to collapse on themselves, one should always pour a gel prior to prepping the samples. Try adding buffer to the top before removing.
10. Remove the comb and pour the buffer to the gel running box. Load ladder and samples in wells. Connect to the power source. Make sure to let the DNA run toward the positive red terminals (Run to Red).