One step 'miniprep' method for the isolation of plasmid DNA - Revision history

Vaishnavi Ananth: /* BioStream version */

2009-11-19T10:02:41Z

BioStream version

←Older revision		Revision as of 10:02, 19 November 2009
Line 26:		Line 26:
	* The largest band appearing after running a gel is usually genomic DNA. I run appropriate control markers.		* The largest band appearing after running a gel is usually genomic DNA. I run appropriate control markers.

-	==~~BioStream~~ version==	+	==BioCoder version==
-	Following is the One step 'miniprep' method for the isolation of plasmid DNA protocol in ~~BioStream~~, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in ~~BioStream~~ (see Source code). More information about ~~BioStream~~ can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]	+	Following is the One step 'miniprep' method for the isolation of plasmid DNA protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
	====Text Output====		====Text Output====
	[[One step 'miniprep' method for the isolation of plasmid DNA protocol]]		[[One step 'miniprep' method for the isolation of plasmid DNA protocol]]

Vaishnavi Ananth at 09:25, 29 September 2009

2009-09-29T09:25:34Z

←Older revision		Revision as of 09:25, 29 September 2009
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	* In step 1, one can pipette 1.5ml of broth spin the microfuge tube, decant 1ml and leave behind 500ul to resuspend the pellet and continue as from step 2. This maximizes the total yield of plasmid.		* In step 1, one can pipette 1.5ml of broth spin the microfuge tube, decant 1ml and leave behind 500ul to resuspend the pellet and continue as from step 2. This maximizes the total yield of plasmid.
	* The largest band appearing after running a gel is usually genomic DNA. I run appropriate control markers.		* The largest band appearing after running a gel is usually genomic DNA. I run appropriate control markers.
		+
		+	==BioStream version==
		+	Following is the One step 'miniprep' method for the isolation of plasmid DNA protocol in BioStream, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
		+	====Text Output====
		+	[[One step 'miniprep' method for the isolation of plasmid DNA protocol]]
		+	====Source Code====
		+	[[One step 'miniprep' method for the isolation of plasmid DNA protocol - source code]]

	[[Category:Protocol]] [[Category:In vitro]] [[Category:Escherichia coli]] [[Category:DNA]]		[[Category:Protocol]] [[Category:In vitro]] [[Category:Escherichia coli]] [[Category:DNA]]

Torsten Waldminghaus at 17:07, 23 February 2009

2009-02-23T17:07:50Z

←Older revision		Revision as of 17:07, 23 February 2009
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		+	{{back to protocols}}
	'''plasmid miniprep'''		'''plasmid miniprep'''

Reshma P. Shetty at 18:49, 9 May 2007

2007-05-09T18:49:36Z

←Older revision		Revision as of 18:49, 9 May 2007
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	* In step 1, one can pipette 1.5ml of broth spin the microfuge tube, decant 1ml and leave behind 500ul to resuspend the pellet and continue as from step 2. This maximizes the total yield of plasmid.		* In step 1, one can pipette 1.5ml of broth spin the microfuge tube, decant 1ml and leave behind 500ul to resuspend the pellet and continue as from step 2. This maximizes the total yield of plasmid.
	* The largest band appearing after running a gel is usually genomic DNA. I run appropriate control markers.		* The largest band appearing after running a gel is usually genomic DNA. I run appropriate control markers.
		+
		+	[[Category:Protocol]] [[Category:In vitro]] [[Category:Escherichia coli]] [[Category:DNA]]

Joseph.borg at 11:39, 24 April 2006

2006-04-24T11:39:57Z

←Older revision		Revision as of 11:39, 24 April 2006
Line 6:		Line 6:

	1. Take 0.5ml of overnight E.coli culture in a microfuge tube. We routinely grow our cells in 'standard 1' bacteriological media supplied by Merck, Germany.		1. Take 0.5ml of overnight E.coli culture in a microfuge tube. We routinely grow our cells in 'standard 1' bacteriological media supplied by Merck, Germany.
		+
	2. Add 0.5ml of phenol:chloroform:isoamylalcohol (25:24:1). The phenol was saturated with TE (10mM Tris, 7.5, 1mM EDTA) prior to mixing with chloroform and isoamylalcohol.		2. Add 0.5ml of phenol:chloroform:isoamylalcohol (25:24:1). The phenol was saturated with TE (10mM Tris, 7.5, 1mM EDTA) prior to mixing with chloroform and isoamylalcohol.
		+
	3. Mix by vortexing at the maximum speed for 1 minute. Alternatively, vortex for 10 seconds and then transfer to eppendorf mixer or an over-the-top rotator for 5 minutes.		3. Mix by vortexing at the maximum speed for 1 minute. Alternatively, vortex for 10 seconds and then transfer to eppendorf mixer or an over-the-top rotator for 5 minutes.
		+
	4. Spin at 12,000g for 5 minutes. During the spin, prepare microfuge tubes with 0.5ml of isopropanol. After the spin, remove carefully about 0.45ml of the upper aqueous phase leaving the interphase undisturbed and add it to the isopropanol. Mix well and spin immediately at 12,000 g for 5 minutes. Addition of salt and cooling is unnecessary.		4. Spin at 12,000g for 5 minutes. During the spin, prepare microfuge tubes with 0.5ml of isopropanol. After the spin, remove carefully about 0.45ml of the upper aqueous phase leaving the interphase undisturbed and add it to the isopropanol. Mix well and spin immediately at 12,000 g for 5 minutes. Addition of salt and cooling is unnecessary.
		+
	5. Pour off the supernatant, add carefully 0.5ml of 70% ethanol to the side of the tube, pour off. Repeat the washing once more. Vacuum dry the pellet and suspend in 100ul/ml RNAse). About 5-10ul of this DNA can now be cleaved with appropriate restriction enzyme(s) for analysis.		5. Pour off the supernatant, add carefully 0.5ml of 70% ethanol to the side of the tube, pour off. Repeat the washing once more. Vacuum dry the pellet and suspend in 100ul/ml RNAse). About 5-10ul of this DNA can now be cleaved with appropriate restriction enzyme(s) for analysis.

Joseph.borg at 11:38, 24 April 2006

2006-04-24T11:38:09Z

New page

'''plasmid miniprep'''

All 'miniprep' methods reported so far for the isolation of plasmid DNA involve multiple pipetting, extraction, centrifugation and changes of minifuge tubes. For screening large number of samples, they are therefore cumbersome, time consuming and not economical.

The technical report below by Chowdhury, K. (1991) is a very fast, simple and one step 'miniprep' procedure. The quality and quantity of DNA obtained by using this procedure is similar to those obtained by the other commonly used procedures of Serghini ''et al.'' (1) or Birboim and Doly (2). According to this procedure, the bacterial culture is directly extracted with a mixture of phenol-chloroform-isoamylalcohol and the liberated DNA is precipitated with isopropanol. This method is now being used routinely in our laboratory for isolating plasmids upto 12kb in size. A detailed description of the method is presented below:

1. Take 0.5ml of overnight E.coli culture in a microfuge tube. We routinely grow our cells in 'standard 1' bacteriological media supplied by Merck, Germany.
2. Add 0.5ml of phenol:chloroform:isoamylalcohol (25:24:1). The phenol was saturated with TE (10mM Tris, 7.5, 1mM EDTA) prior to mixing with chloroform and isoamylalcohol.
3. Mix by vortexing at the maximum speed for 1 minute. Alternatively, vortex for 10 seconds and then transfer to eppendorf mixer or an over-the-top rotator for 5 minutes.
4. Spin at 12,000g for 5 minutes. During the spin, prepare microfuge tubes with 0.5ml of isopropanol. After the spin, remove carefully about 0.45ml of the upper aqueous phase leaving the interphase undisturbed and add it to the isopropanol. Mix well and spin immediately at 12,000 g for 5 minutes. Addition of salt and cooling is unnecessary.
5. Pour off the supernatant, add carefully 0.5ml of 70% ethanol to the side of the tube, pour off. Repeat the washing once more. Vacuum dry the pellet and suspend in 100ul/ml RNAse). About 5-10ul of this DNA can now be cleaved with appropriate restriction enzyme(s) for analysis.

References
* Chowdhury, K. (1991) One step 'miniprep' method for the isolation of plasmid DNA. ''Nucl. Acids Res'' '''19''':10 2792
* Serghini, M.A. Ritzenthaler, C., and Pinck, J. (1989) ''Nucl. Acids Res'' 17, 3604
* Birnboim, H.C., and Doly, J. (1979) ''Nucl. Acids Res.'' 13, 1513 - 1523.

Additional Notes
* Sterile LB broth works very well in this protocol
* In step 1, one can pipette 1.5ml of broth spin the microfuge tube, decant 1ml and leave behind 500ul to resuspend the pellet and continue as from step 2. This maximizes the total yield of plasmid.
* The largest band appearing after running a gel is usually genomic DNA. I run appropriate control markers.