Add up to 50 mg of plant tissue to the 2 mL homogenization tubes that contain the grinding bead (large bead) and solid phase extraction matrix (small beads). Then, add 500 uL of lysis buffer. (This step has already been done for you)
Place the homogenization tube into the Genogrinder and homogenize the sample for 4 minutes at 1500 rpm. When samples are completely homogenized, the tube will lack foam. If the plant sample is not completely homogenized, repeat the homogenization process in 2 minute intervals until complete.
What is the main point of the lysis step? Sketch what your sample looks like.:
|
|
|
|
|
|
|
Centrifuge the homogenization tubes at 15,000 x g for 5 minutes to pellet the debris, grinding resin, and contaminants.
Transfer the clear supernatant into a clean, labeled tube.
What is the main point of this centrifugation step and what is in the supernatant?:
|
|
|
|
|
|
|
Sketch your sample and label the DNA, RNA, proteins, and other cellular debris:
|
|
|
|
|
|
|
What is the main point of adding the RNAse?:
|
|
|
|
|
|
|
Determine the volume of solution in your tube. Add 7/10 volume of 100% isopropanol to your tube. (In other words, find 70% of the volume of your current solution and add that amount of isoproponal to your tube. Error on the high side, if your volume estimations are fuzzy.)
Vortex your tube for 2-3 seconds. Incubate at -20C for 15 minutes.
Transfer the solution to a spin column place in a collection tube. Centrifuge the column at 8,000 x g for 1 minute to bind the DNA to the column.
What is the main point of the isopropanol precipitation step? Where is the DNA?:
|
|
|
|
|
|
|
Wash the column with 250 uL of ice cold 70% ethanol. Centrifuge the column a 8,000 x g for 1 minute to pass through the wash solution.
Repeat step 9.
What is the main point of the wash step? Where is the DNA?:
|
|
|
|
|
|
|
Elute the DNA by placing the column in a clean collection tube and by adding 100 uL of water. Centrifuge the column at 15,000 x g for 1 minute.
Repeat step 11, but note that the second elution will contain less yield.
What is the main point of the elution step? Where is the DNA?:
|
|
|
|
|
|
|