Steps:
1) Weigh out 50g of diced onion (see Appendix - The Balance). Transfer to 250 ml beaker.
2) Add 100 ml of homogenizing medium and incubate in 60° C water bath for 15 minutes.
3) Cool mixture in ice bath to 20° C.
4) Homogenize in blender for several 3 - 5 second intervals at low speed.
5) Pour into 1000 ml beaker and place in ice bath for 15-20 min.
6) Filter the mixture through 4 layers of cheesecloth into a 250 ml beaker. Leave the foam behind.
7) Dispose of your onion remains in the trash. DO NOT LEAVE ONIONS IN THE SINK!!! Deproteinization. During this process the chromosomal proteins are stripped from the DNA. They are further denatured and precipitated from the mixture, leaving the DNA behind.
7) Pour 50 ml of your homogenate into a 125 Erlenmeyer ml flask.
8) The instructor will add 2 ml of Chloroform to the homogenate with a 5 ml pipette.
9) Swirl the flask gently.
10) Pour the homogenate into another 125 Erlenmeyer flask. Leave the chloroform and the protein layers behind on the bottom. Rinse the first flask carefully and use again.
11) After the deproteinization, pour the homogenate into a 125 Erlenmeyer flask. Decant, making sure there are no proteins or chloroform in the homogenate even at the risk of leaving some homogenate behind.
12) Pour the waste chloroform into a labeled waste bottle on the side bench. Do not pour down the drain. Precipitation. DNA does not stay in solution in ETOH. DNA should precipitate out of the solution in a thick white stringy mass, which can be spooled out with a glass rod. 13) Place beaker with the homogenate in an ice bath and cool until it reaches 10° - 15° C.
14) Slowly add ice-cold ETOH down the side of the beaker.
15) Spool out and wind the stringy DNA on a glass rod by rotating the rod in one direction in the beaker.
Materials:
