Hop DNA Isolation

Hop DNA Extraction Protocol

1. Obtain an adequate amount (specify) 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 3.3 ml of buffer per gram of wet (16 ml per gram of dried) hop leaves, and incubate for 1 to 4 hours at 60-65°C.
4. Transfer 900 μl into fresh tube and add 600 μl of 24:1 CHCl₃:octanol to each and shake.
5. Centrifuge at 5000g for 10 minutes.
6. Remove supernatant and put in new vials (800 μl in each).
7. Add 5μl of RNAase in each vial and incubate at 37°C for 30 minutes.
8. Remove supernatant (discard) and add 0.5 ml of buffer wash solution to each pellet and allowed to sit until needed.
9. Dry pellet and add 100 μl ddH₂O and incubate at 45°C until needed.
10. Run electrophoresis for analysis.

Prepared solutions

1. Buffer: 100 ml: 50 mM Tris/HCl (ph 8.0), 1.8 M NaCl, 50 mM EDTA. Then add 10 mg/ml of CTAB ( 200 mg per 20 ml buffer, final conc. = 1%) and 1 μl/ml 2-mercaptoethanol (20 μl to 20 ml buffer; final conc. = 0.1%).
2. Wash buffer 100 ml: 200 μl 5M NH4OAc (final conc. = 10 mM), 76.0 ml abs. ethanol (final conc. = 76%), and 23.8 ml of sterilized water.