Hop DNA Isolation: Difference between revisions
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==Hop DNA Extraction Protocol== | ==Hop DNA Extraction Protocol== | ||
#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 (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. | #Assume 90% of mass is water weight. | ||
#Add 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. | ||
#Put 900μl into 6 tubes and add 600μl of 24:1 CHCl<sub>3</sub>:octanol to each and shake. | #Put 900 μl into 6 tubes and add 600 μl 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 (800μl in each). | #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. | #Add 5μl of RNAase in each vial and incubate at 37°C for 30 minutes. | ||
#Remove supernatant (discard) and add 0. | #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 100μl ddH<sub>2</sub>O and incubate at 45°C until needed. | #Dry pellet and add 100 μl ddH<sub>2</sub>O and incubate at 45°C until needed. | ||
#Run electrophoresis for analysis. | #Run electrophoresis for analysis. | ||
==Prepared solutions== | ==Prepared solutions== | ||
#Buffer: | #Buffer: 100 ml: 50 mM Tris/HCl (ph 8.0), 1.8 M NaCl, 50 mM EDTA. Then add 200 mg/ml of CTAB and 20 μl/ml 2-mercaptoethanol. | ||
#Buffer wash solution: | #Buffer wash solution: 5 ml: 20 μl 5M NH4OAc, 3.8 ml absolute ethanol, and 1.18 ml of sterilized water. | ||
[[Category:Protocol]] [[Category:In vitro]] [[Category:Plant]] | [[Category:Protocol]] [[Category:In vitro]] [[Category:Plant]] | ||
Revision as of 11:24, 18 January 2008
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.
Prepared solutions
- Buffer: 100 ml: 50 mM Tris/HCl (ph 8.0), 1.8 M NaCl, 50 mM EDTA. Then add 200 mg/ml of CTAB and 20 μl/ml 2-mercaptoethanol.
- Buffer wash solution: 5 ml: 20 μl 5M NH4OAc, 3.8 ml absolute ethanol, and 1.18 ml of sterilized water.
