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.
- Transfer 900 μl into fresh tube
- add 600 μl of 24:1 CHCl3:octanol and invert gently (do NOT vortex!).
- Centrifuge at 5000g for 10 minutes.
- Transfer supernatant (800 μl) into new 2-ml tube.
- Add 5μl of RNAase and incubate at 37°C for 30 minutes (or more).
- Add 0.6 volumes Isopropanol and mix gently by inverting the tubes. Check for DNA precipitation.
- Spin down for 10 min.
- Add 500 μl wash buffer and incubate 10 min. at RT.
- Carefully remove wash buffer. Don't lose DNA pellet!
- Briefly centrifuge to collect pellet at bottom of tube - remove any remaining wash buffer.
- Dry pellet at RT or 50°C to speed up.
- Add 100 μl ddH2O to dissolve DNA.
- Store at -20°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.