Hop DNA Isolation

Hop DNA Extraction Protocol

 * 1) Obtain an adequate amount (~ 1g) 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&deg;C.
 * 4) Transfer 900 &mu;l into fresh tube
 * 5) add 600 &mu;l of 24:1 CHCl3:octanol and invert gently (do NOT vortex!).
 * 6) Centrifuge at 5000g for 10 minutes.
 * 7) Transfer supernatant (800 &mu;l) into new 2-ml tube.
 * 8) Add 5&mu;l of RNAase and incubate at 37&deg;C for 30 minutes (or more).
 * 9) Add 0.6 volumes Isopropanol and mix gently by inverting the tubes. Check for DNA precipitation.
 * 10) Spin down for 10 min. at RT.
 * 11) Add 500 &mu;l wash buffer and incubate 10 min. at RT.
 * 12) Carefully remove wash buffer. Don't lose DNA pellet!
 * 13) Briefly centrifuge to collect pellet at bottom of tube - remove any remaining wash buffer.
 * 14) Dry pellet at RT or 50&deg;C to speed up.
 * 15) Add 100 &mu;l ddH2O to dissolve DNA.
 * 16) Store at -20&deg;C until needed.
 * 17) 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 &mu;l/ml 2-mercaptoethanol (20 &mu;l to 20 ml buffer; final conc. = 0.1%).
 * 2) Wash buffer 100 ml: 200 &mu;l 5M NH4OAc (final conc. = 10 mM), 76.0 ml abs. ethanol (final conc. = 76%), and 23.8 ml of sterilized water.