Hop DNA Isolation - Revision history

Axel at 16:52, 20 April 2008

2008-04-20T16:52:33Z

←Older revision		Revision as of 16:52, 20 April 2008
Line 9:		Line 9:
	#Add 5μl of RNAase and incubate at 37°C for 30 minutes (or more).		#Add 5μl of RNAase and incubate at 37°C for 30 minutes (or more).
	#Add 0.6 volumes Isopropanol and mix gently by inverting the tubes. Check for DNA precipitation.		#Add 0.6 volumes Isopropanol and mix gently by inverting the tubes. Check for DNA precipitation.
-	#Spin down for 10 min.	+	#Spin down for 10 min. at RT.
	#Add 500 μl wash buffer and incubate 10 min. at RT.		#Add 500 μl wash buffer and incubate 10 min. at RT.
	#Carefully remove wash buffer. Don't lose DNA pellet!		#Carefully remove wash buffer. Don't lose DNA pellet!

Axel at 16:51, 20 April 2008

2008-04-20T16:51:35Z

←Older revision		Revision as of 16:51, 20 April 2008
Line 1:		Line 1:
	==Hop DNA Extraction Protocol==		==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).	+	#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.
	#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.		#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.

Axel at 18:55, 18 January 2008

2008-01-18T18:55:41Z

←Older revision		Revision as of 18:55, 18 January 2008
Line 3:		Line 3:
	#Assume 90% of mass is water weight.		#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.		#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.
-	#Transfer 900 μl into fresh tube ~~and~~ add 600 μl of 24:1 CHCl<sub>3</sub>:octanol ~~to each~~ and ~~shake~~.	+	#Transfer 900 μl into fresh tube
		+	#add 600 μl 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.
-	#~~Remove~~ supernatant ~~and put in new vials~~ (800 μl ~~in each~~).	+	#Transfer supernatant (800 μl) into new 2-ml tube.
-	#Add 5μl of RNAase ~~in each vial~~ and incubate at 37°C for 30 minutes.	+	#Add 5μl of RNAase and incubate at 37°C for 30 minutes (or more).
-	#~~Remove supernatant (discard) and add~~ 0.~~5 ml of~~ buffer wash ~~solution to each~~ pellet ~~and allowed~~ to ~~sit until needed~~.	+	#Add 0.6 volumes Isopropanol and mix gently by inverting the tubes. Check for DNA precipitation.
-	#Dry pellet ~~and add~~ 100 μl ddH<sub>2</sub>O ~~and incubate~~ at 45°C until needed.	+	#Spin down for 10 min.
		+	#Add 500 μl wash buffer and incubate 10 min. at RT.
		+	#Carefully remove wash buffer. Don't lose DNA pellet!
		+	#Briefly centrifuge to collect pellet at bottom of tube - remove any remaining wash buffer.
		+	#Dry pellet at RT or 50°C to speed up.
		+	#Add 100 μl ddH<sub>2</sub>O to dissolve DNA.
		+	#Store at -20°C until needed.
	#Run electrophoresis for analysis.		#Run electrophoresis for analysis.

Axel at 18:48, 18 January 2008

2008-01-18T18:48:13Z

←Older revision		Revision as of 18:48, 18 January 2008
Line 3:		Line 3:
	#Assume 90% of mass is water weight.		#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.		#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 CHCl<sub>3</sub>:octanol to each and shake.	+	#Transfer 900 μl into fresh tube 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 (800 μl in each).		#Remove supernatant and put in new vials (800 μl in each).

Axel at 18:47, 18 January 2008

2008-01-18T18:47:03Z

←Older revision		Revision as of 18:47, 18 January 2008
Line 14:		Line 14:

	#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%).		#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%).
-	#~~Buffer wash solution: 5~~ ml: 20 μl 5M NH4OAc, 3.8 ml ~~absolute~~ ethanol, and 1.18 ml of sterilized water.	+	#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.

	[[Category:Protocol]] [[Category:In vitro]] [[Category:Plant]]		[[Category:Protocol]] [[Category:In vitro]] [[Category:Plant]]

Axel at 18:44, 18 January 2008

2008-01-18T18:44:48Z

←Older revision		Revision as of 18:44, 18 January 2008
Line 13:		Line 13:
	==Prepared solutions==		==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: 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%).
	#Buffer wash solution: 5 ml: 20 μl 5M NH4OAc, 3.8 ml absolute ethanol, and 1.18 ml of sterilized water.		#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]]

Axel at 18:24, 18 January 2008

2008-01-18T18:24:33Z

Reshma P. Shetty at 17:48, 9 May 2007

2007-05-09T17:48:12Z

←Older revision		Revision as of 17:48, 9 May 2007
Line 15:		Line 15:
	#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: 5ml: 20μl 5M NH4OAc, 3.8ml absolute ethanol, and 1.18ml of sterilized water.		#Buffer wash solution: 5ml: 20μl 5M NH4OAc, 3.8ml absolute ethanol, and 1.18ml of sterilized water.
		+
		+	[[Category:Protocol]] [[Category:In vitro]] [[Category:Plant]]

Axel: /* Hop DNA Extraction Protocol */

2007-04-09T20:00:15Z

Hop DNA Extraction Protocol

←Older revision		Revision as of 20:00, 9 April 2007
Line 2:		Line 2:
	#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 900μl into 6 tubes and add 600μl of 24:1 CHCl<sub>3</sub>:octanol to each and shake.		#Put 900μl into 6 tubes and add 600μl 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 (800μl in each).
-	#Add 5μl of RNAase in each vial and incubate at 37 ~~degrees Celsius~~ for 30 minutes.	+	#Add 5μl of RNAase in each vial and incubate at 37°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 100μl ddH<sub>2</sub>O and incubate at 45 ~~degrees Celsius~~ until needed.	+	#Dry pellet and add 100μl ddH<sub>2</sub>O and incubate at 45°C until needed.
	#Run electrophoresis for analysis.		#Run electrophoresis for analysis.

Axel: /* Hop DNA Extraction Protocol */

2007-04-09T19:59:29Z

Hop DNA Extraction Protocol

←Older revision		Revision as of 19:59, 9 April 2007
Line 3:		Line 3:
	#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 60-65C.
-	#Put 900μl into 6 tubes and add 600μl of 24:1 ~~CHCl3~~:octanol to each and shake.	+	#Put 900μl into 6 tubes and add 600μl 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 (800μl in each).

←Older revision		Revision as of 18:24, 18 January 2008
Line 1:		Line 1:
	==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.~~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.	+	#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.~~5ml~~ of buffer wash solution to each pellet and allowed to sit until needed.	+	#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: ~~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: 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: ~~5ml~~: 20μl 5M NH4OAc, 3.~~8ml~~ absolute ethanol, and 1.~~18ml~~ of sterilized water.	+	#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]]