Hop DNA Isolation: Difference between revisions
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#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 (800μl in each). | ||
#Add 5μl of RNAase in each vial. | #Add 5μl of RNAase in each vial and incubate at 37 degrees Celsius for 30 minutes. | ||
# | #Remove supernatant (discard) and add 0.5ml of buffer wash solution to each pellet and allowed to sit until needed. | ||
#Dry pellet and add 100μl and incubate at 44 degrees Celsius until needed. | |||
#Run electrophoresis for analysis. (10μl of sample with 2μl of loading dye) | |||
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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 μl/ml 2-mercaptoethanol. | 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. | ||
Buffer wash solution: | Buffer wash solution: 5ml: 20μl 5M NH4OAc, 3.8ml absolute ethanol, and 1.19ml of sterilized water. | ||
Revision as of 11:56, 26 March 2007
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).
- 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.
- Put 900μl into 6 tubes and add 600μl of 24:1 CHCl3:octanol to each and shake.
- Centrifuge at 5000g for 10 minutes.
- Remove supernatant and put in new vials (800μl in each).
- Add 5μl of RNAase in each vial and incubate at 37 degrees Celsius for 30 minutes.
- Remove supernatant (discard) and add 0.5ml of buffer wash solution to each pellet and allowed to sit until needed.
- Dry pellet and add 100μl and incubate at 44 degrees Celsius until needed.
- Run electrophoresis for analysis. (10μl of sample with 2μl of loading dye)
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.
Buffer wash solution: 5ml: 20μl 5M NH4OAc, 3.8ml absolute ethanol, and 1.19ml of sterilized water.
