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Protocol/6
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Revision as of 00:34, 26 October 2010
Protocol 6: Agarose Gel Electrophoresis
What you will need:
- 1X TAE
- Graduated cylinder
- 250 mL flask
- Agarose
- Gel forming tray
- Ethidium bromide
Before you start:
- know that ETHIDIUM BROMIDE (EtBr) IS A MUTAGEN!!! Always handle it separately from everything else, with gloves on. Any EtBr waste (including gloves and tips) must go into the yellow bucket provided at the EtBr station. Any used gels must also go into this bucket.
- UV LIGHT DAMAGES YOUR DNA, SKIN, AND EYES!!! Always wear protective eyewear when working with it.
Procedure:
Making a 1% gel:
- Dilute stock of 50X TAE to 1X with ddH2O.
- Weigh out enough agarose to make 1% gel. (1% of 70 mL is 0.70 g)
- Transfer agarose to flask containing 1X TAE. Form an improvised cap by inverting a 50 ml beaker into the neck of larger flask.
- Melt agarose in microwave, stirring ever 15-20 seconds. This should take about 2 min.
- Allow agarose solution to cool until flask can be handled comfortably.
- Pour agarose into Gel Mold and insert comb
- Allow to solidify completely. While the gel is solidifying, prepare the samples.
- Pull comb out of gel and place the gel along with its stand into a gel electrophoresis box.
- Pour 1X TAE over gel so that gel is covered by 3-5 mm of buffer.
- Load samples into lane, our wells hold about 15uL. Make sure the samples have been mixed with 10X Loading Dye to a final concentration of 1X. * Do not forget to load 1kb+ ladder into one of the lanes.
Running a gel:
- VERY IMPORTANT: Hook electrodes to gel apparatus. Nucleic acids are negatively charged, so they will run to the positive (red) terminal. Set the power source accordingly.
- Turn on the power source. You can run at a voltage of your choice, but make sure you watch the bands so they don't run off. For instance, at 100 V it could take from 30 to 45 mins can vary.
Staining:
- Place gel in Ethidium Bromide container, and set the rotator to a fairly low speed. Leave it there for about five minutes.
- Take gel out and put it on the tray with the spatula.
Imaging:
- Carry gel to dark room with being cautious to prevent contaminating the environment with EtBr.
- ie. Use your gloved hand to put the gel in the imager. Use your ungloved hand to close the door. Set the dial to white epi light.
- Image using the Kodak program and imager.
