Anthocyanin Extraction Protocol:
(250-500 mg tissue/sample in 2 ml screw cap tube)
1. Add 1 mL of 2N HCl to sample and let soak overnight.
2. Spin down and pull off super-natant into a 2 mL screw-cap tube.
3. Put tubes into boiling bath for 30-40 minutes. Let cool.
4. Add 1/2 volume (~450-ish ul) Ethyl Acetate (organics cabinet). Vortex!
5. Spin at 10,000 rpm for 1 minute. Discard supernatant.
6. Repeat steps 4 and 5 two more times.
7. Put in 50 degree oven for about 10 minutes or until remaining organic layer has evaporated. Keep oven door cracked.
8. Add 100-150 Iso-Amyl Alcohol. Vortex hard and spin down. Put supernatant con-taining anthocyanins into .5 ml tube.
9. Repeat step 8 if the bottom layer still had lots of color.
10. Dry samples in oven until just a pellet remains. This will take a while. As it is drying, a bubble of crap will form at the bottom. Get rid of this.
11. Resuspend in MeOH 1% HCl.
12. Spin the sample really hard. Put the sample into a new tube.