Process of Extraction of Anthocyanins from Eggplant
1) Using tweezers remove individual plants from the soil and remove the roots with scissors. Measure the weight of each individual and place in a 1.5 mL microfuge tube. Place this tube directly into liquid N2 to freeze plant tissue. 2) Remove tubes from the liquid N2 and quickly grind the plant tissue with a pestle. Add 300 L of Methanol 1%HCl and continue to grind the plant tissue. Allow extraction to occur overnight in a dark refrigerator.
3) Add 200 L Milli-Q H20 to each tube. Next, go to the fume hood and add 500 L of chloroform to each tube. Spin the tubes in a centrifuge set at the highest rpm for 2-5 minutes.
4) Return to the fume hood and extract the supernatant/aqueous (top) solution from the tube using a pipetmann. To do this start by setting the pipetmann to 400 L. Slightly tilt the tube to one side, place the tip of the pipetmann just below the surface and remove the supernatant while moving the tip toward the bottom of the tube. Place the supernatant in a new 1.5 mL microfuge tube. Be careful not to remove any white plant tissue or organic (bottom) solution.
5) Bring the volume up to 800 L by adding 400L of a 60% Methanol 1% HCl : 40% Milli-Q H20 solution to each tube. For example, if you have 60 tubes you need 24 mL of the 60% 40% solution, therefore mix 14.4 mL Methanol 1%HCl and 9.6 mL Milli-Q H20 then add 400L of this solution to each tube. Remember to make a little extra 60%40% solution so you don’t run out on the last tube!
6) Read the absorbance of each tube at 530nm and 657nm using the spectrophotometer or plate reader. The blank should be 480L Methanol 1%HCl and 320L Milli-Q H20 for a total of
3) Add 200 L Milli-Q H20 to each tube. Next, go to the fume hood and add 500 L of chloroform to each tube. Spin the tubes in a centrifuge set at the highest rpm for 2-5 minutes.
4) Return to the fume hood and extract the supernatant/aqueous (top) solution from the tube using a pipetmann. To do this start by setting the pipetmann to 400 L. Slightly tilt the tube to one side, place the tip of the pipetmann just below the surface and remove the supernatant while moving the tip toward the bottom of the tube. Place the supernatant in a new 1.5 mL microfuge tube. Be careful not to remove any white plant tissue or organic (bottom) solution.
5) Bring the volume up to 800 L by adding 400L of a 60% Methanol 1% HCl : 40% Milli-Q H20 solution to each tube. For example, if you have 60 tubes you need 24 mL of the 60% 40% solution, therefore mix 14.4 mL Methanol 1%HCl and 9.6 mL Milli-Q H20 then add 400L of this solution to each tube. Remember to make a little extra 60%40% solution so you don’t run out on the last tube!
6) Read the absorbance of each tube at 530nm and 657nm using the spectrophotometer or plate reader. The blank should be 480L Methanol 1%HCl and 320L Milli-Q H20 for a total of