Hop DNA Isolation

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(Hop DNA Extraction Protocol)
Current revision (11:52, 20 April 2008) (view source)
 
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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).
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#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).
#Assume 90% of mass is water weight.
#Assume 90% of mass is water weight.
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#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.
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#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.
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#Put 900μl into 6 tubes and add 600μl of 24:1 CHCl3:octanol to each and shake.
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#Transfer 900 μl into fresh tube
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#add 600 &mu;l of 24:1 CHCl<sub>3</sub>:octanol and invert gently (do NOT vortex!).
#Centrifuge at 5000g for 10 minutes.
#Centrifuge at 5000g for 10 minutes.
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#Remove supernatant and put in new vials (800&mu;l in each).
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#Transfer supernatant (800 &mu;l) into new 2-ml tube.
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#Add 5&mu;l of RNAase in each vial and incubate at 37 degrees Celsius for 30 minutes.
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#Add 5&mu;l of RNAase and incubate at 37&deg;C for 30 minutes (or more).
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#Remove supernatant (discard) and add 0.5ml of buffer wash solution to each pellet and allowed to sit until needed.
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#Add 0.6 volumes Isopropanol and mix gently by inverting the tubes. Check for DNA precipitation.
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#Dry pellet and add 100&mu;l and incubate at 44 degrees Celsius until needed.
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#Spin down for 10 min. at RT.
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#Run electrophoresis for analysis. (10&mu;l of sample with 2&mu;l of loading dye) 
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#Add 500 &mu;l wash buffer and incubate 10 min. at RT.
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#Carefully remove wash buffer. Don't lose DNA pellet!
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#Briefly centrifuge to collect pellet at bottom of tube - remove any remaining wash buffer.
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#Dry pellet at RT or 50&deg;C to speed up.
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#Add 100 &mu;l ddH<sub>2</sub>O to dissolve DNA.
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#Store at -20&deg;C until needed.
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#Run electrophoresis for analysis.
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==Prepared solutions==
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#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%).
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#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.
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[[Category:Protocol]] [[Category:In vitro]] [[Category:Plant]]
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Buffer: 100ml: tris/HCl (ph=8.0) 50mM, NaCl 1.8M, EDTA 50mM.  Then add 200mg/ml of CTAB and 20 &mu;l/ml 2-mercaptoethanol.
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Buffer wash solution: 5ml: 20&mu;l 5M NH4OAc, 3.8ml absolute ethanol, and 1.19ml of sterilized water.
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Current revision

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°C.
  4. Transfer 900 μl into fresh tube
  5. add 600 μl of 24:1 CHCl3:octanol and invert gently (do NOT vortex!).
  6. Centrifuge at 5000g for 10 minutes.
  7. Transfer supernatant (800 μl) into new 2-ml tube.
  8. Add 5μl of RNAase and incubate at 37°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 μl 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°C to speed up.
  15. Add 100 μl ddH2O to dissolve DNA.
  16. Store at -20°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 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.
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