Agarose Gel Electrophoresis and DNA Fragment Purification

[domba]

Supplies:

1. Micropipetter and tips
2. Microcentrifuge and tubes
3. Gel apparatus
4. Power supply for the gel apparatus
5. Polaroid(R) setup (with proper filter - SYBR Green/Gold gel stain photographic filter) and UV light box Teacher's note: UV is dangerous to the skin and eyes, so supervision is very important.
6. Microwave

Procedures:

1. Set up 1 % (w/v) agarose gel:
* Add 0.5 g agarose powder to a flask.
* Add 50 ml TAE buffer to make enough for the gel apparatus.
* Heat (by microwave) briefly to dissolve the powder.Teacher's note: Do not allow it to boil over, and use protection since the flask is hot.
* Cool to 50-60 degrees C.
* Pour into the gel tray.
* Lower the comb (to form wells) gently into the molten gel.
* Allow the gel to solidify in the refrigerator for 15 minutes.Teacher's note: Care should be taken to make sure the gel is leveled while solidifying.
2. Prepare DNA marker:
* Take 3 ul of DNA marker stock solution into a tube.
* Add 22 ul of distilled water.
* Add 5 ul of 6 X loading dye and mix. (observation: contents in the tubes change color from the addition of loading dye.)
3. Add 4 ul of 6 X loading dye into 20 ul of samples for gel electrophoresis. (observation: contents in the tubes change color from the addition of loading dye.)Teacher's note: The amount of DNA used varies with the purpose of the agarose gel electrophoresis.
4. Spin for 2 seconds in a microcentrifuge.
5. Put solidified gel into the gel apparatus.
6. Add TAE buffer to cover the gel and remove the comb.
7. Load 10 ul from each tube into a well.
8. Load 10 ul of DNA marker into a well.
9. Run the gel at 100-150 V for 20 minutes. (observation: dye front moves toward the positive charge.)Teacher's note: High voltage is used here, so supervision is very important. Use 100 V with mini-gel apparatus, and use 150 V with midi-gel apparatus.
10. Staine the gel for 30 minutes with a 1:10,000 dilution of SYBR Green I(R) nucleic acid gel stain.
11. Put on proper face shield and any other necessary gear to protect skin and eyes from UV.
12. Move the gel onto UV light box.
13. Take a Polaroid photo under UV.Teacher's note: Supervision is very important. UV is damaging skin, eyes, and also to the DNA. Turn on UV for brief moments only.
14. If necessary, use a spatula to cut out DNA band(s) of interest under UV, transfer into a tube, and store at -20 degrees C.Teacher's note: Supervision is very important. UV is damaging skin, eyes, and also to the DNA. Turn on UV for brief moments only.