Hop DNA Isolation

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(→Hop DNA Extraction Protocol)
(→Hop DNA Extraction Protocol)
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#Obtain an adequate amount of fresh hop leaves and crush them with liquid Nitrogen and a small amount of Carborundum powder (fine 320 grit).
#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.
#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.
+
#Add 3.3ml of buffer per gram of wet (16ml per gram of dried) hop leaves, and incubate for 1 to 4 hours at 60-65°C.
#Put 900&mu;l into 6 tubes and add 600&mu;l of 24:1 CHCl<sub>3</sub>:octanol to each and shake.
#Put 900&mu;l into 6 tubes and add 600&mu;l of 24:1 CHCl<sub>3</sub>:octanol to each and shake.
#Centrifuge at 5000g for 10 minutes.
#Centrifuge at 5000g for 10 minutes.
#Remove supernatant and put in new vials (800&mu;l in each).
#Remove supernatant and put in new vials (800&mu;l in each).
-
#Add 5&mu;l of RNAase in each vial and incubate at 37 degrees Celsius for 30 minutes.
+
#Add 5&mu;l of RNAase in each vial and incubate at 37&deg;C for 30 minutes.
#Remove supernatant (discard) and add 0.5ml of buffer wash solution to each pellet and allowed to sit until needed.
#Remove supernatant (discard) and add 0.5ml of buffer wash solution to each pellet and allowed to sit until needed.
-
#Dry pellet and add 100&mu;l ddH<sub>2</sub>O and incubate at 45 degrees Celsius until needed.
+
#Dry pellet and add 100&mu;l ddH<sub>2</sub>O and incubate at 45&deg;C until needed.
#Run electrophoresis for analysis.
#Run electrophoresis for analysis.

Revision as of 16:00, 9 April 2007

Hop DNA Extraction Protocol

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

Prepared solutions

  1. 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.
  2. Buffer wash solution: 5ml: 20μl 5M NH4OAc, 3.8ml absolute ethanol, and 1.18ml of sterilized water.
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