Richard Lab:Agarose Gel Electrophoresis
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The consensus electrophoresis protocol should be consulted if deviating from the procedure outlined here.
Procedure
- Place the gel tray perpendicular in the electrophoresis to create a casting bay.
- Wipe the tray clean with a Kim-wipe and level the tray using the bubble level.
- Weigh out the desired amount of agarose and add it to 100ml of 1X TBE in a 300ml flask.
- Microwave the flask on high for 80 seconds. Be sure that it does not boil over.
- Swirl the microwaved agarose in the flask until the solution becomes clear.
- Pour the melted solution into the casting bay and insert the comb.
- Prepare the DNA ladder by combining the following:
- 10ul DNA ladder
- 1ul SYBR green (100X)
- 1ul Bromophenol Blue
- Prepare the DNA Samples by combining the following:
- 40ul DNA preparation
- 4ul SYBR Green (100X)
- 4ul Bromophenol Blue
- Remove the comb from the cured gel and realign the gel in the chamber.
- Adjust the buffer level by adding 1X TBE to the chamber until the buffer just covers the gel. The buffer can be reused a few times.
- Pipette the samples into the wells
- Apply 150 volts and run for approximately 60 minutes.
- Photograph the gel using the UVP transilluminator system.
The amount of agarose to use in your gel depends on the DNA in question. As a rough guide: Agarose (g/100mL) 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
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