Mesoplasma florum:Genomic DNA - Revision history

Tk: /* Qiagen Genomic Tip protocol */

2009-11-11T16:13:01Z

Qiagen Genomic Tip protocol

←Older revision		Revision as of 16:13, 11 November 2009
Line 58:		Line 58:
	* Pellet cells at 5000 x g for 10 minutes		* Pellet cells at 5000 x g for 10 minutes
	* Resuspend the pellet in 11 ml of buffer B1 (with RNAse)		* Resuspend the pellet in 11 ml of buffer B1 (with RNAse)
-	* Add 500 μl of proteinase-K ~~solutio~~, 20 mg/ml (10 mg)	+	* Add 500 μl of proteinase-K solution, 20 mg/ml (10 mg)
	* incubate at 37°C for at least 30 minutes		* incubate at 37°C for at least 30 minutes
	* Add 4 ml of buffer B2 and mix or vortex briefly		* Add 4 ml of buffer B2 and mix or vortex briefly

Tk: /* Qiagen Genomic Tip protocol */

2009-11-11T16:08:40Z

Qiagen Genomic Tip protocol

←Older revision		Revision as of 16:08, 11 November 2009
Line 81:		Line 81:
	* Centrifuge at 5000 x g for 15 minutes at 4°C, wash with 70% ethanol		* Centrifuge at 5000 x g for 15 minutes at 4°C, wash with 70% ethanol
	* dissolve in 100-200 μl of TE pH 8.0 on a shaker at 55°C or overnight		* dissolve in 100-200 μl of TE pH 8.0 on a shaker at 55°C or overnight
		+
		+
		+	* For diagnosis of problems:
		+	** precipitate the DNA from the samples 1-4 taken above with 0.7 volumes of isopropanol
		+	** wash with 70% ethanol
		+	** Resuspend in 20 μl TE
		+	** Run 10 μl on a 0.5% agarose gel


	[[category:Mesoplasma florum]][[category:protocol]]		[[category:Mesoplasma florum]][[category:protocol]]

Tk at 16:03, 11 November 2009

2009-11-11T16:03:55Z

←Older revision		Revision as of 16:03, 11 November 2009
Line 44:		Line 44:

	A critical element is the NaCl concentration prior to adding CTAB. With < 500 mM NaCl, DNA precipitates. Above that point, polysaccharides and proteins precipitate, but the DNA stays in solution.		A critical element is the NaCl concentration prior to adding CTAB. With < 500 mM NaCl, DNA precipitates. Above that point, polysaccharides and proteins precipitate, but the DNA stays in solution.
		+
		+
		+	==Reference==
		+	* PMID 7433111
		+	* This protocol follows closely the bacterial genomic DNA protocol from Current Protocols section 2.4, contributed by Kate Wilson.


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	* Centrifuge at 5000 x g for 15 minutes at 4°C, wash with 70% ethanol		* Centrifuge at 5000 x g for 15 minutes at 4°C, wash with 70% ethanol
	* dissolve in 100-200 μl of TE pH 8.0 on a shaker at 55°C or overnight		* dissolve in 100-200 μl of TE pH 8.0 on a shaker at 55°C or overnight
-
-	~~==Reference==~~
-	* PMID 7433111
-	* This protocol follows closely the bacterial genomic DNA protocol from Current Protocols section 2.4, contributed by Kate Wilson.
-


	[[category:Mesoplasma florum]][[category:protocol]]		[[category:Mesoplasma florum]][[category:protocol]]

Tk: /* Protocol */

2009-11-10T20:01:10Z

Protocol

←Older revision		Revision as of 20:01, 10 November 2009
Line 18:		Line 18:
	* 70% ethanol		* 70% ethanol

-	==Protocol==	+	==CTAB Protocol==

	* Culture 10 ml of medium 1161 infected with Mesoplasma florum in a 15 ml centrifuge tubes at 30° without shaking.		* Culture 10 ml of medium 1161 infected with Mesoplasma florum in a 15 ml centrifuge tubes at 30° without shaking.
Line 45:		Line 45:
	A critical element is the NaCl concentration prior to adding CTAB. With < 500 mM NaCl, DNA precipitates. Above that point, polysaccharides and proteins precipitate, but the DNA stays in solution.		A critical element is the NaCl concentration prior to adding CTAB. With < 500 mM NaCl, DNA precipitates. Above that point, polysaccharides and proteins precipitate, but the DNA stays in solution.

		+
		+	==Qiagen Genomic Tip protocol==
		+
		+	For buffers, see [[Qiagen Buffers]].
		+
		+	* Grow 40 ml cultures of the Mesoplasma species at 30°C in 1161 medium.
		+	* Pellet cells at 5000 x g for 10 minutes
		+	* Resuspend the pellet in 11 ml of buffer B1 (with RNAse)
		+	* Add 500 μl of proteinase-K solutio, 20 mg/ml (10 mg)
		+	* incubate at 37°C for at least 30 minutes
		+	* Add 4 ml of buffer B2 and mix or vortex briefly
		+	* Incubate at 50°C for 30 minutes; the lysate should become clear
		+	* If necessary, pellet particulates
		+	* Save a 300 μl sample 1
		+
		+	* Equilibrate a genomic tip 500/G with 10 ml of buffer QBT and allow flow through
		+	* Vortex the sample for 10-20 seconds to reduce viscosity
		+	* Sample should flow through, or can be diluted with buffer QBT before loading
		+	* limit flow to 20-40 drops/min under pressure
		+	* save a 1200 μl sample of the flow through -- sample 2
		+
		+	* Wash the column with 15 ml of buffer QC twice or more
		+	* save a 600 μl sample of the flow through -- sample 3
		+
		+	* Elute with 15 ml of buffer QF prewarmed to 50°C
		+	* save a 600 μl sample of the elution -- sample 4
		+
		+	* precipitate DNA by adding 10.5 ml (0.7 volumes) of isopropanol
		+	* invert the tube several times and recover by spooling on a glass rod or
		+	* Centrifuge at 5000 x g for 15 minutes at 4°C, wash with 70% ethanol
		+	* dissolve in 100-200 μl of TE pH 8.0 on a shaker at 55°C or overnight

	==Reference==		==Reference==

Torsten Waldminghaus at 17:15, 23 February 2009

2009-02-23T17:15:39Z

←Older revision		Revision as of 17:15, 23 February 2009
Line 1:		Line 1:
		+	{{back to protocols}}
	Extraction of genomic DNA from Mesoplasma florum		Extraction of genomic DNA from Mesoplasma florum

Tk at 19:20, 9 September 2007

2007-09-09T19:20:39Z

←Older revision		Revision as of 19:20, 9 September 2007
Line 1:		Line 1:
	Extraction of genomic DNA from Mesoplasma florum		Extraction of genomic DNA from Mesoplasma florum
-
-	~~This protocol follows closely the bacterial genomic DNA protocol from Current Protocols section 2.4, contributed by Kate Wilson.~~
-

	==Materials==		==Materials==

Tk at 19:20, 9 September 2007

2007-09-09T19:20:24Z

←Older revision		Revision as of 19:20, 9 September 2007
Line 4:		Line 4:


-	Materials:	+	==Materials==

	* ATCC 1161 culture medium		* ATCC 1161 culture medium
Line 20:		Line 20:
	* 70% ethanol		* 70% ethanol

		+	==Protocol==

-	Culture 10 ml of medium 1161 infected with Mesoplasma florum in a 15 ml centrifuge tubes at 30° without shaking.	+	* Culture 10 ml of medium 1161 infected with Mesoplasma florum in a 15 ml centrifuge tubes at 30° without shaking.
		+	* Transfer 2x 1.8 ml into two 2 ml tubes. Spin down at 17000g for 2 minutes and retain the pellet.
		+	* Resuspend the pellet by vortexing first, then adding 567 μl of TE.
		+	* Add 3 μl of proteinase-K 20 mg/ml and 30 μl of SDS 10% solution, mix and incubate at 37° for 1 hour
		+	* Add 100 μl of 5 M NaCl and mix (critical before adding CTAB)
		+	* Add 80 μl of CTAB / NaCl solution, mix
		+	* Incubate 10 minutes at 65°
		+	* Add an equal volume (800 μl) of chloroform / isoamyl alcohol 24:1, vortex and spin down at 17000g for 2 minutes
		+	* Setup fresh 2 ml tubes with 800 μl of phenol / chloroform / isoamyl alcohol 25:24:1, transfer the supernatent, and mix carefully.
		+	* Spin down at 17000g for 2 minutes
		+	* Setup fresh 2 ml tubes with 800 μl of chloroform / isoamyl alcohol 24:1, transfer the supernatent, and mix carefully.
		+	* Spin down at 17000g for 2 minutes
		+	* Setup fresh 1.5 ml tubes loaded with 1 μl Novagen Pellet Paint NF and transfer the supernatent into those tubes.
		+	* Add 0.6 volumes of isopropanol and chill in the -80 freezer for 30 minutes
		+	* Spin down at 17000g for 20 minutes and remove the supernatent carefully retaining the blue pellet.
		+	* Fill the tube with 70% ethanol to wash, and spin down at 17000g for 2 minutes.
		+	* Remove the ethanol carefully leaving the blue pellet. This is best done by using a 1 ml pipet to remove the bulk of the ethanol, then a 10 μl tip to remove the remainder. Spinning the tube down after most of the ethanol is removed will assist in removing the last 10-20 μl.
		+	* Let the tube air dry for 1/2 hour until the ethanol odor is no longer present.
		+	* Redissolve the DNA pellet in 100 μl TE (this will take an hour or so).
		+	* Spin down the tube briefly and measure OD and 260/280 ratios.
		+	* Expect around 500 ng/μl concentration (total 50 μg).

-	~~Transfer 2x 1.8 ml into two 2 ml tubes. Spin down at 17000g for 2 minutes and retain the pellet.~~

-	~~Resuspend~~ the ~~pellet by vortexing first, then~~ adding ~~567 μl of TE~~.	+	A critical element is the NaCl concentration prior to adding CTAB. With < 500 mM NaCl, DNA precipitates. Above that point, polysaccharides and proteins precipitate, but the DNA stays in solution.

-	~~Add 3 μl of proteinase-K 20 mg/ml and 30 μl of SDS 10% solution, mix and incubate at 37° for 1 hour~~

-	~~Add 100 μl of 5 M NaCl and mix (critical before adding CTAB)~~	+	==Reference==
		+	* PMID 7433111
		+	* This protocol follows closely the bacterial genomic DNA protocol from Current Protocols section 2.4, contributed by Kate Wilson.

-	~~Add 80 μl of CTAB / NaCl solution, mix~~
-
-	~~Incubate 10 minutes at 65°~~
-
-	~~Add an equal volume (800 μl) of chloroform / isoamyl alcohol 24:1, vortex and spin down at 17000g for 2 minutes~~
-
-	~~Setup fresh 2 ml tubes with 800 μl of phenol / chloroform / isoamyl alcohol 25:24:1, transfer the supernatent, and mix carefully.~~
-
-	~~Spin down at 17000g for 2 minutes~~
-
-	~~Setup fresh 2 ml tubes with 800 μl of chloroform / isoamyl alcohol 24:1, transfer the supernatent, and mix carefully.~~
-
-	~~Spin down at 17000g for 2 minutes~~
-
-	~~Setup fresh 1.5 ml tubes loaded with 1 μl Novagen Pellet Paint NF and transfer the supernatent into those tubes.~~
-
-	~~Add 0.6 volumes of isopropanol and chill in the -80 freezer for 30 minutes~~
-
-	~~Spin down at 17000g for 20 minutes and remove the supernatent carefully retaining the blue pellet.~~
-
-	~~Fill the tube with 70% ethanol to wash, and spin down at 17000g for 2 minutes.~~
-
-	Remove the ethanol carefully leaving the blue pellet. This is best done by using a 1 ml pipet to remove the bulk of the ethanol, then a 10 μl tip to remove the remainder. Spinning the tube down after most of the ethanol is removed will assist in removing the last 10-20 μl. Let the tube air dry for 1/2 hour until the ethanol odor is no longer present.
-
-	~~Redissolve the DNA pellet in 100 μl TE (this will take an hour or so).~~
-
-	~~Spin down the tube briefly and measure OD and 260/280 ratios.~~
-
-	~~Expect around 500 ng/μl concentration (total 50 μg).~~
-
-
-	~~A critical element is the NaCl concentration prior to adding CTAB. With < 500 mM NaCl, DNA precipitates. Above that point, polysaccharides and proteins precipitate, but the DNA stays in solution.~~

-	~~Literature reference: PMID 7433111~~

	[[category:Mesoplasma florum]][[category:protocol]]		[[category:Mesoplasma florum]][[category:protocol]]

Tk at 19:16, 9 September 2007

2007-09-09T19:16:11Z

←Older revision		Revision as of 19:16, 9 September 2007
Line 45:		Line 45:
	Spin down at 17000g for 2 minutes		Spin down at 17000g for 2 minutes

-	~~Remove the supernatent to a~~ fresh ~~tube~~ 1.5 ml ~~tube and add~~ 1 μl of Novagen Pellet Paint NF~~, followed by 0.6 volumes of isopropanol; DNA should precipitate as a clear/white pearly substance in solution~~.	+	Setup fresh 1.5 ml tubes loaded with 1 μl Novagen Pellet Paint NF and transfer the supernatent into those tubes.

-	Spin down at 17000g for 2 minutes and remove the supernatent carefully retaining the blue pellet.	+	Add 0.6 volumes of isopropanol and chill in the -80 freezer for 30 minutes
		+
		+	Spin down at 17000g for 20 minutes and remove the supernatent carefully retaining the blue pellet.

	Fill the tube with 70% ethanol to wash, and spin down at 17000g for 2 minutes.		Fill the tube with 70% ethanol to wash, and spin down at 17000g for 2 minutes.

Tk at 04:55, 4 September 2007

2007-09-04T04:55:55Z

←Older revision		Revision as of 04:55, 4 September 2007
Line 35:		Line 35:
	Incubate 10 minutes at 65°		Incubate 10 minutes at 65°

-	Add an equal volume (800 μl) of chloroform / isoamyl alcohol 24:1, vortex and spin down at 17000g for 6 minutes	+	Add an equal volume (800 μl) of chloroform / isoamyl alcohol 24:1, vortex and spin down at 17000g for 2 minutes

	Setup fresh 2 ml tubes with 800 μl of phenol / chloroform / isoamyl alcohol 25:24:1, transfer the supernatent, and mix carefully.		Setup fresh 2 ml tubes with 800 μl of phenol / chloroform / isoamyl alcohol 25:24:1, transfer the supernatent, and mix carefully.
Line 45:		Line 45:
	Spin down at 17000g for 2 minutes		Spin down at 17000g for 2 minutes

-	Remove the supernatent to a fresh tube 1.5 ml tube and add 1 μl of Novagen Pellet Paint NF, followed by 0.6 volumes of isopropanol; DNA should precipitate as a clear/white pearly substance in solution~~. Spin down at high speed and pour off the supernatent carefully retaining the blue pellet~~.	+	Remove the supernatent to a fresh tube 1.5 ml tube and add 1 μl of Novagen Pellet Paint NF, followed by 0.6 volumes of isopropanol; DNA should precipitate as a clear/white pearly substance in solution.

-	Fill the tube with 70% ethanol to wash, and spin down at 17000g for 6 minutes.	+	Spin down at 17000g for 2 minutes and remove the supernatent carefully retaining the blue pellet.
		+
		+	Fill the tube with 70% ethanol to wash, and spin down at 17000g for 2 minutes.

	Remove the ethanol carefully leaving the blue pellet. This is best done by using a 1 ml pipet to remove the bulk of the ethanol, then a 10 μl tip to remove the remainder. Spinning the tube down after most of the ethanol is removed will assist in removing the last 10-20 μl. Let the tube air dry for 1/2 hour until the ethanol odor is no longer present.		Remove the ethanol carefully leaving the blue pellet. This is best done by using a 1 ml pipet to remove the bulk of the ethanol, then a 10 μl tip to remove the remainder. Spinning the tube down after most of the ethanol is removed will assist in removing the last 10-20 μl. Let the tube air dry for 1/2 hour until the ethanol odor is no longer present.

Tk at 04:53, 4 September 2007

2007-09-04T04:53:59Z

←Older revision		Revision as of 04:53, 4 September 2007
Line 23:		Line 23:
	Culture 10 ml of medium 1161 infected with Mesoplasma florum in a 15 ml centrifuge tubes at 30° without shaking.		Culture 10 ml of medium 1161 infected with Mesoplasma florum in a 15 ml centrifuge tubes at 30° without shaking.

-	Transfer 2x 1.8 ml into two 2 ml ~~eppendorfs~~. Spin down at ~~8000g~~ for 2 minutes and retain the pellet.	+	Transfer 2x 1.8 ml into two 2 ml tubes. Spin down at 17000g for 2 minutes and retain the pellet.

	Resuspend the pellet by vortexing first, then adding 567 μl of TE.		Resuspend the pellet by vortexing first, then adding 567 μl of TE.
Line 35:		Line 35:
	Incubate 10 minutes at 65°		Incubate 10 minutes at 65°

-	Add an equal volume (800 μl) of chloroform / isoamyl alcohol 24:1, vortex and spin down at ~~7000g~~ for 6 minutes	+	Add an equal volume (800 μl) of chloroform / isoamyl alcohol 24:1, vortex and spin down at 17000g for 6 minutes

-	~~Remove the supernatent to a~~ fresh ~~tube and add an equal volume~~ of phenol / chloroform / isoamyl alcohol 25:24:1 and mix carefully.	+	Setup fresh 2 ml tubes with 800 μl of phenol / chloroform / isoamyl alcohol 25:24:1, transfer the supernatent, and mix carefully.

	Spin down at 17000g for 2 minutes		Spin down at 17000g for 2 minutes

-	~~Remove the supernatent to a~~ fresh ~~tube and add an equal volume~~ of chloroform / isoamyl alcohol 24:1 and mix carefully.	+	Setup fresh 2 ml tubes with 800 μl of chloroform / isoamyl alcohol 24:1, transfer the supernatent, and mix carefully.

	Spin down at 17000g for 2 minutes		Spin down at 17000g for 2 minutes

-	Remove the supernatent to a fresh tube and add 1 μl of Novagen Pellet Paint NF, followed by 0.6 volumes of isopropanol; DNA should precipitate as a clear/white pearly substance in solution. Spin down at high speed and pour off the supernatent carefully retaining the blue pellet.	+	Remove the supernatent to a fresh tube 1.5 ml tube and add 1 μl of Novagen Pellet Paint NF, followed by 0.6 volumes of isopropanol; DNA should precipitate as a clear/white pearly substance in solution. Spin down at high speed and pour off the supernatent carefully retaining the blue pellet.

	Fill the tube with 70% ethanol to wash, and spin down at 17000g for 6 minutes.		Fill the tube with 70% ethanol to wash, and spin down at 17000g for 6 minutes.
Line 54:		Line 54:

	Spin down the tube briefly and measure OD and 260/280 ratios.		Spin down the tube briefly and measure OD and 260/280 ratios.
		+
		+	Expect around 500 ng/μl concentration (total 50 μg).