Streptomyces:Protocols/Mini-Maxi Prep - Revision history

Vaishnavi Ananth: /* BioStream version */

2009-11-19T09:59:36Z

BioStream version

←Older revision		Revision as of 09:59, 19 November 2009
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-	==~~BioStream~~ version==	+	==BioCoder version==
-	Following is the Small Scale Plasmid Isolation (Mini-Maxi prep) 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 Small Scale Plasmid Isolation (Mini-Maxi prep) 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====
	[[Small Scale Plasmid Isolation (Miniprep) protocol]]<br/>		[[Small Scale Plasmid Isolation (Miniprep) protocol]]<br/>

Vaishnavi Ananth at 07:52, 23 October 2009

2009-10-23T07:52:19Z

←Older revision		Revision as of 07:52, 23 October 2009
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	==BioStream version==		==BioStream version==
-	Following is the Small Scale Plasmid Isolation (~~Miniprep~~) 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 Small Scale Plasmid Isolation (Mini-Maxi prep) 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====		====Text Output====
-	[[Small Scale Plasmid Isolation (Miniprep) protocol]]	+	[[Small Scale Plasmid Isolation (Miniprep) protocol]]<br/>
	[[Small Scale Plasmid Isolation (Maxiprep) protocol]]		[[Small Scale Plasmid Isolation (Maxiprep) protocol]]
	====Source Code====		====Source Code====
-	[[Small Scale Plasmid Isolation (Miniprep) protocol - source code]]	+	[[Small Scale Plasmid Isolation (Miniprep) protocol - source code]]<br/>
	[[Small Scale Plasmid Isolation (Maxiprep) protocol - source code]]		[[Small Scale Plasmid Isolation (Maxiprep) protocol - source code]]

Vaishnavi Ananth at 07:51, 23 October 2009

2009-10-23T07:51:21Z

←Older revision		Revision as of 07:51, 23 October 2009
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	====Text Output====		====Text Output====
	[[Small Scale Plasmid Isolation (Miniprep) protocol]]		[[Small Scale Plasmid Isolation (Miniprep) protocol]]
		+	[[Small Scale Plasmid Isolation (Maxiprep) protocol]]
	====Source Code====		====Source Code====
	[[Small Scale Plasmid Isolation (Miniprep) protocol - source code]]		[[Small Scale Plasmid Isolation (Miniprep) protocol - source code]]
		+	[[Small Scale Plasmid Isolation (Maxiprep) protocol - source code]]

Vaishnavi Ananth at 07:37, 23 October 2009

2009-10-23T07:37:05Z

←Older revision		Revision as of 07:37, 23 October 2009
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	<!--CONTENT END-->		<!--CONTENT END-->
	\|}<br style="clear:both" />		\|}<br style="clear:both" />
		+
		+	==BioStream version==
		+	Following is the Small Scale Plasmid Isolation (Miniprep) 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====
		+	[[Small Scale Plasmid Isolation (Miniprep) protocol]]
		+	====Source Code====
		+	[[Small Scale Plasmid Isolation (Miniprep) protocol - source code]]

Torsten Waldminghaus at 14:23, 9 December 2008

2008-12-09T14:23:35Z

←Older revision		Revision as of 14:23, 9 December 2008
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	[[Category:Protocol]]		[[Category:Protocol]]
-	[[Category:~~Escherichia coli~~]]	+	[[Category:Streptomyces]]
	<!--CONTENT START-->		<!--CONTENT START-->

Streptomyces at 13:00, 14 August 2007

2007-08-14T13:00:48Z

←Older revision		Revision as of 13:00, 14 August 2007
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	{{Streptomyces}}		{{Streptomyces}}
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Streptomyces at 10:04, 10 August 2007

2007-08-10T10:04:47Z

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	<!--THESE CATEGORY LINES ADD THE PROTOCOL TO THE OWW PROTOCOLS PAGE-->		<!--THESE CATEGORY LINES ADD THE PROTOCOL TO THE OWW PROTOCOLS PAGE-->
	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:Escherichia coli]]		[[Category:Escherichia coli]]
		+	<!--CONTENT START-->

	==Small Scale Plasmid Isolation (Mini / Maxi Prep)==		==Small Scale Plasmid Isolation (Mini / Maxi Prep)==

Streptomyces at 10:03, 10 August 2007

2007-08-10T10:03:35Z

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	<!--CONTENT START-->		<!--CONTENT START-->
		+	<!--THESE CATEGORY LINES ADD THE PROTOCOL TO THE OWW PROTOCOLS PAGE-->
		+	[[Category:Protocol]]
		+	[[Category:Escherichia coli]]

	==Small Scale Plasmid Isolation (Mini / Maxi Prep)==		==Small Scale Plasmid Isolation (Mini / Maxi Prep)==

Streptomyces at 08:36, 10 August 2007

2007-08-10T08:36:32Z

←Older revision		Revision as of 08:36, 10 August 2007
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	\| \|\| Phenol/Chloroform extraction \|\| ~10 minutes		\| \|\| Phenol/Chloroform extraction \|\| ~10 minutes
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		+	\| colspan="3"\|
	\|-		\|-
	\| Optional: \|\| RNase & repeated Phenol/Chloroform \|\| ~45 minutes		\| Optional: \|\| RNase & repeated Phenol/Chloroform \|\| ~45 minutes
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		+	\| colspan="3"\|
	\|-		\|-
	\| \|\| Alcohol precipitation \|\| ~25 minutes		\| \|\| Alcohol precipitation \|\| ~25 minutes

Streptomyces: New page: ==Protocols - Mini / Maxi Prep== {{Streptomyces}}
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2007-06-14T10:02:21Z

New page: ==Protocols - Mini / Maxi Prep==  {{Streptomyces}} {|cellspacing="5" cellpadding="10" style="background:#000; width: 800px;" |-valign="top" |style="ba...

New page

==Protocols - Mini / Maxi Prep==

{{Streptomyces}}
 
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==Small Scale Plasmid Isolation (Mini / Maxi Prep)==

'''Description'''
 
A mini prep / maxi prep is used to isolate plasmid or cosmid DNA from bacteria, normally E.coli. An overnight culture of cells is lysed and the majority of the cells contents are removed with the DNA remaining dissolved in solution. Phenol/Chloroform extraction is used to purify the DNA contaminated by histones and other soluble proteins. Equal volumes of a phenol:chloroform mixture and the aqueous DNA sample are mixed, forming a biphasic mixture. The proteins partition into the organic phase while the DNA (as well as other contaminants such as salts, sugars, etc.) remain in the aqueous phase. This (top) phase is removed and the DNA precipitated with alcohol; all anionic salts remain in the aqueous liquid. Washing with 70% ethanol removes the last of the contaminants leaving the final product.

 
{| cellspacing="0" cellpadding="0"
|-
| style="width: 150px;"| Approx. Duration: || style="width: 225px;"| || style="width: 75px;"| ||
|-
| || Preparation || ~10 minutes
|-
| || Cell Lysis || ~35 minutes
|-
| || Phenol/Chloroform extraction || ~10 minutes
|-
| Optional: || RNase & repeated Phenol/Chloroform || ~45 minutes
|-
| || Alcohol precipitation || ~25 minutes
|-
| || Washing, drying & re-suspending || ~30 minutes
|-
| || Whole protocol || ~2 hours 35 minutes
|}

'''Uses'''
 
Extract large quantities of good quality plasmid DNA for further work, e.g. sub-cloning, transformations, verification by digestion/sequencing, PCR, southern blots etc…

'''Prerequisites'''
 
An overnight culture needs to be grown of the bacterial strain containing the required plasmid/cosmid to be extracted. A fume cupboard dedicated to phenol/chloroform work & waste needs to be available.

'''Safety'''
 
COSHH forms need to be created for the lab in which the work is done. All users need to understand the risks associated with both phenol & chloroform. A phenol decontamination kit (PEG300:Meths) must be located near the fume hood in which the work is done. General laboratory & molecular microbiology safety rules apply.

'''Requirements'''
 
Cells, ice, 15mL falcon tube(s), micro-centrifuge tube(s) (MCT), Solution I, Solution II, Solution III, Phenol, Chloroform, isopropanol (propan-2-ol), 70% ethanol, sdH2O (sterile distilled water), falcon centrifuge @ ~4°C, micro-centrifuge @ ~4°C.
 
Optional: incubator (i.e. a water bath) @ 37°C, Ribonuclease A (RNase).

'''Preparation'''
 
Chill Solution I and Solution III on ice; the isopropanol and 70% ethanol should be placed in the freezer. Make fresh Solution II (see notes). Label the falcons or MCTs. Ensure the correct sized centrifuge is set to ~4°C.

'''Method'''
 
As the cells will be lysed and DNA extracted there is no requirement for sterility. All steps should be performed chilled (on ice) except for the phenol/chloroform step(s) and the RNase incubation step (obviously!). Spins should be done at the maximum capacity of the centrifuge/tubes.
A mini prep uses 3mL of cells; a '''maxi prep''' uses 50mL of cells ('''different steps are indicated in bold''').
 
 
Cell lysis:
# Take the overnight culture and spin down 1.5mL ('''50mL''') of cells for 3 ('''5''') minutes.
# Pour off the supernatant and add another 1.5mL of cells, spin.
# Re-suspend the pellet in 100µL ('''1mL''') of ice cold Solution I.
# Add 200µL ('''2mL''') of Solution II and ''gently invert'' the tube ('''''roll the falcon''''') to mix. Leave on ice for 5 minutes.
# Add 150µL ('''1.5mL''') of Solution III and shake vigorously. Leave on ice for 10 minutes.
# Centrifuge for 5 minutes.
 
Phenol/Chloroform Cleanup:
# Transfer the supernatant by pipetting to a clean MCT ('''15mL falcon''').
# Add 0.5 volumes of phenol and 0.5 volumes of chloroform to 1 volume of supernatant ~250:250->500µL ('''2.5:2.5->5mL''').
# Mix well by vortexing and centrifuge for 5 minutes.
# Carefully take the top aqueous phase and transfer to a new tube.
 
RNase (optional):
# Add RNase to a final concentration of 3µg/mL (5µL of 3mg/mL diluted stock / '''5µL of 30mg/mL stock''').
# Incubate in a 37°C water bath for 30-45 minutes.
# Repeat the phenol/chloroform step.
 
Alcohol Precipitation / Purification:
# Add an equal volume of ice cold isopropanol to the aqueous solution.
# Mix well by inversion and leave on ice for 10 minutes.
# Centrifuge at maximum speed for 15 minutes.
# Carefully pour off the isopropanol leaving the DNA pellet.
# Wash the pellet with 200µL ('''2mL''') of 70% ethanol by pipetting.
# Centrifuge for 5 minutes.
# Remove all the alcohol by pipetting and air dry the pellet for up to 30 minutes.
# Re-suspend the DNA pellet in 30-50µL ('''200-300µL''') sdH2O.
# Store the samples at -20°C.

'''Notes'''
 
Solution I: 50mM Tris•HCl pH8.0 / 10mM EDTA pH8.0
 
Solution II: 200mM NaOH / 1% SDS
 
Solution III: 3M KOAc (Potassium Acetate) pH5.5

After re-suspending the cells in Solution I the mixture should be homogenous. The addition of Solution II causes a translucent goo of lysed cell matter to be observed. Solution III precipitates the majority of this by instant decrease in pH.
 
Solution II contains sodium hydroxide which is critical in lysing the cells. Ideally it needs to be made fresh or at least from a fresh solution of sodium hydroxide. This is because the sodium hydroxide absorbs carbon dioxide from the atmosphere causing it to change into sodium carbonate, which will not lyse the cells. After the addition of Solution II gentle mixing is required so the DNA is not damaged. The DNA will have various (relatively large) proteins bound and anything more than gentle inversion or rolling the falcon can shear the DNA.
 
Phenol is toxic and should be handled with extreme care. Its solution is buffered with 3% NaCl, which forms an upper biphasic layer. When using the phenol ensure you pipette from below this salt buffer layer. When removing the top aqueous phase after treating with phenol/chloroform be careful not to disturb the lower organic layer. Any phenol taken with the aqueous phase may degrade the RNase or affect later reactions, such as PCR.
 
If the alcohol solutions are ice cold and the samples centrifuged @ ~4°C the DNA pellet will become more visible and therefore, easier to work with. Also, the yield may be slightly higher.
 
The addition of RNase is a worthwhile step, especially for maxi preps. It makes the analysis of future reactions easier, for example, the viewing of small fragments after a restriction digest.


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