Hop DNA Isolation
Hop DNA Extraction Protocol
- Obtain an adequate amount of fresh hop leaves and crush them with liquid Nitrogen and a small amount of Carborundum powder (fine 320 grit).
- Assume 90% of mass is water weight.
- 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.
- Put 900μl into 6 tubes and add 600μl of 24:1 CHCl3:octanol to each and shake.
- Centrifuge at 5000g for 10 minutes.
- Remove supernatant and put in new vials (800μl in each).
- Add 5μl of RNAase in each vial and incubate at 37 degrees Celsius for 30 minutes.
- Remove supernatant (discard) and add 0.5ml of buffer wash solution to each pellet and allowed to sit until needed.
- Dry pellet and add 100μl ddH2O and incubate at 45 degrees Celsius until needed.
- Run electrophoresis for analysis.
- 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.
- Buffer wash solution: 5ml: 20μl 5M NH4OAc, 3.8ml absolute ethanol, and 1.18ml of sterilized water.