Team:Washington/Protocols/Optogenetics/LightAppExperiments
From 2012.igem.org
General Protocol for Light App Experiments
Overnight cultures
- Cast a gel
- Place it in gel box in running buffer
- Load samples
- Run the gel
- 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:
Agarose Concentration (g/100mL) | Optimal DNA Resolution (kb) |
---|---|
0.5 | 1 - 30 |
0.7 | 0.8 - 12 |
1.0 | 0.5 - 10 |
1.2 | 0.4 - 7 |
1.5 | 0.2 - 3 |
- Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer (see the documentation for your gelbox -- 50mL makes a good, thick gel for a 7x10cm gelbox).
- Microwave until the agarose is fully melted. This depends strongly on your microwave, but a 90 seconds at full power or 3 minutes at half power seem to provide decent results. As long as you do not burn the agarose and nothing bubbles over, this step is robust.
- From here on, a heat protective glove should be used any time the heated flask must be touched!
- Let the agarose cool on the bench for ~5 minutes.
- At this point add your DNA visualization reagent, e.g., ethidium bromide at a volume appropriate for the amount of melted agarose. This amount will depend on the concentration of the stock solution of the stain.
- The flask will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles, and should therefore be periodically swirled while the agarose is cooling.
- Pour the agarose solution into the gel casting apparatus. A pipette tip should be used to pop or shove to the side any bubbles.
- After bubbles have been popped, the comb should be inserted, and the gel should be allowed to cool for about 30 minutes, until solid.
- Because removal of the comb too early can cause the wells to collapse on themselves, one should always poor a gel prior to prepping the samples.
Adapted thanks to [http://openwetware.org/wiki/Agarose_gel_electrophoresis OpenWetWare protocols].