Hop DNA Isolation

Hop DNA Extraction Protocol

1. Obtain an adequate amount of fresh hop leaves and crush them with liquid Nitrogen and a small amount of Carborundum powder (fine 320 grit).
2. Assume 90% of mass is water weight.
3. Add 3.3ml of buffer per gram of wet (16ml per gram of dried) hop leaves, and incubate for 1 to 4 hours at 60-65C.
4. Put 900μl into 6 tubes and add 600μl of 24:1 CHCl3:octanol to each and shake.
5. Centrifuge at 5000g for 10 minutes.
6. Remove supernatant and put in new vials (800μl in each).
7. Add 5μl of RNAase in each vial and incubate at 37 degrees Celsius for 30 minutes.
8. Remove supernatant (discard) and add 0.5ml of buffer wash solution to each pellet and allowed to sit until needed.
9. Dry pellet and add 100μl and incubate at 44 degrees Celsius until needed.
10. Run electrophoresis for analysis. (10μl of sample with 2μl of loading dye)

Prepared solutions

1. Buffer: 100ml: tris/HCl (ph=8.0) 50mM, NaCl 1.8M, EDTA 50mM. Then add 200mg/ml of CTAB and 20 μl/ml 2-mercaptoethanol.
2. Buffer wash solution: 5ml: 20μl 5M NH4OAc, 3.8ml absolute ethanol, and 1.19ml of sterilized water.