Collect Tissue for DNA: http://www.youtube.com/watch?v=UYUmWeIaWpQ
1. Place a piece of leaf tissue of approximately 1 cm2 in a mortar and pestle. Add 600 µl of Lysis Buffer and carefully* grind the tissue until liquefied.
Video: http://www.youtube.com/watch?v=RNFT-VM05V4
* Note: When grinding, be very careful not to introduce froth into the homogenate as this will make it very difficult to collect it for the next step. Grind with smooth motions, bringing the pestle across the entire bottom of the mortar before lifting up. The lysis buffer contains detergent so it is easy for it to get frothy.
2. Collect 600 ul of homogenate and place it in a labeled 1.5 ml microtube. If you don’t recover 600 ul of homogenate, add more Lysis Buffer to the Mortar and pestle, grind briefly, collect liquid and add to the homogenate in the tube until the total recovered volume equals 600 ul.
3. Perform an organic extraction:
- a. In the fume hood, add 600 ul chloroform and close the cap tightly. While holding the cap closed, shake vigorously to form an emulsion. Video: http://www.youtube.com/watch?v=urmyFrIpp2Q
- b. Incubate at room temperature for 5 minutes. During this incubation, mix periodically to keep the mixture emulsified.
- c. Centrifuge at 12,000 x g for 5 min. at room temperature. After centrifugation, you should see two distinct phases and a layer of solid material between them. The upper liquid phase is the aqueous phase and contains the DNA.
- d. Collect the aqueous (top) phase without disturbing the material at the interface. Try to collect the entire aqueous phase. (The DNA is in the aqueous phase.) Video: http://www.youtube.com/watch?v=7W3zEnupemk
5. Incubate 5 minutes at room temperature.
6. Place tubes in the centrifuge with the hinge of each tube toward the outside of the rotor and centrifuge at 5,000 x g for 5 min. You should see a small, but visible pellet at the bottom. By placing the hinge of the tube to the outside during centrifugation, you can figure out where to look for the pellet. (Think of how the tube is positioned in the rotor and thus where the pellet should form.
7. Remove and discard the supernatant. (The DNA is in the pellet.)
8. Wash the pellet with 70% ethanol.
- a. Add 500 ul of 70% ethanol to each pellet.
- b. Vortex to dislodge pellet. Don’t worry; the DNA will not redissolve in 70% ethanol.
- c. Centrifuge 5,000 x g for 5 min. Remove supernatant
- d. Centrifuge at 5,000 x g for 20 seconds to get all liquid to the bottom of the tube and remove all liquid with a micropipette.
9. Air dry the pellet by leaving the tube open on the bench top. This usually takes less than 10 minutes. However, the criteria is dryness, not time.
10. Redissolve the DNA that is in the pellet by adding 40 ul of TE buffer.
11. Incubate at 55 degrees for 15 min. During this time, mix every 5 minutes.
12. Centrifuge at 10,000 x g for 5 minutes. Collect all of the supernatant and transfer to a new labeled 0.5 ml microtube. Discard the pellet
Store the DNA in the refrigerator.
