Hop DNA Isolation
Hop DNA Extraction Protocol
- 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).
- Assume 90% of mass is water weight.
- 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.
- Put 900 μl into 6 tubes and add 600 μl of 24:1 CHCl3:octanol to each and shake.
- Centrifuge at 5000g for 10 minutes.
- Remove supernatant and put in new vials (800 μl in each).
- Add 5μl of RNAase in each vial and incubate at 37°C for 30 minutes.
- Remove supernatant (discard) and add 0.5 ml of buffer wash solution to each pellet and allowed to sit until needed.
- Dry pellet and add 100 μl ddH2O and incubate at 45°C until needed.
- Run electrophoresis for analysis.
- 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%).
- 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.