Protocols/Create - Revision history

Anitha Bodepudi: /* Miniprep */

Anitha Bodepudi — Tue, 19 Mar 2013 01:50:31 GMT

Miniprep

←Older revision		Revision as of 01:50, 19 March 2013
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	==Miniprep==		==Miniprep==

-	This ~~get~~ you the DNA	+	This gets you the DNA
-		+
-		+

	==Procedure==		==Procedure==

Radoslaw Kwasniak: /* Procedure */

Radoslaw Kwasniak — Thu, 12 Jul 2012 18:33:10 GMT

Procedure

←Older revision		Revision as of 18:33, 12 July 2012
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	==Procedure==		==Procedure==
-	~~# In~~ a ~~PCR tube, mix the components on ice in the order they are listed above.~~	+	Collect a turbid sample of 10ml overnight growth
-	# ~~Perform thermocycling program~~	+	# Centrifuge universal bottle for 10min 2600rpm
-	## ~~95 °C 5 min~~	+	# Discard supernatant
-	## ~~95 °C 30 s~~	+	# Resuspend cell pellets with 250μL of P1 Buffer by pipetting
-	## ~~T<sub>H</sub> 30 s~~	+	# Transfer to sterile labelled eppendorf tube
-	## ~~72 °C~~ 1 ~~min~~ for ~~each~~ 1 ~~kb PCR product~~	+	# Add 250μL of P2 Lysing Buffer
-	## ~~Repeat steps 2-4 a total of 12-36 times~~ (~~24 is standard~~).	+	# Wait for 3-5 minutes (e.g. 3.5min)
-	## ~~72 °C 5 min~~	+	# Add 350μL of N3 Neutralisation Buffer - title well to ensure mixing (10-12 times)
-	## ~~12 °C hold~~	+	# Centrifuge sample at 13K rpm for 10 minutes
		+	# Add supernatent to a nickle filter (ensure not solids)
		+	# Centrifuge for 1 minute (13K rpm) and discard flowthrough
		+	# Add 500μL PB to wash filter
		+	# Centrifuge for 1 minute (13K rpm) and discard flowthrough
		+	# Add 750μL PE (with Ethanol) for final wash
		+	# Centrifuge for 1 minute (13K rpm) and discard flowthrough
		+	# Centrifuge for 1 additional minute (13K rpm) and discard flowthrough
		+	# Transfer filter to sterile labelled eppendorf tube
		+	# Add 30μL Nuclease free water to centre of column
		+	# Wait for 1 minute
		+	# Centrifuge for 1 minute (13K rpm) to elute
		+	# Store sample on ice or in -20°C

	==Notes==		==Notes==

Radoslaw Kwasniak: /* Materials */

Radoslaw Kwasniak — Thu, 12 Jul 2012 18:21:03 GMT

Materials

←Older revision		Revision as of 18:21, 12 July 2012
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	This get you the DNA		This get you the DNA

-	~~==Materials==~~

-	~~For a 50 μL PCR reaction:~~
-
-	* 35 μL H<sub>2</sub>O
-	* 5 μL 10X PCR buffer
-	* 5 μL 2mM dNTPs (each)
-	* 1.5 μL 50mM MgCl<sub>2</sub>
-	* 1 μL 50μM sense primer
-	* 1 μL 50μM antisense primer
-	* 1 μL 5nM DNA template
-	* 0.5 μL TAQ DNA polyermerase

	==Procedure==		==Procedure==

Radoslaw Kwasniak: /* Overview */

Radoslaw Kwasniak — Thu, 12 Jul 2012 18:20:47 GMT

Overview

←Older revision		Revision as of 18:20, 12 July 2012
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-	==~~Overview~~==	+	==Miniprep==

-	~~Replace this sentence with a brief description of~~ the ~~protocol and its goal.~~	+	This get you the DNA

	==Materials==		==Materials==

Radoslaw Kwasniak: /* Creating a New Protocol */

Radoslaw Kwasniak — Thu, 12 Jul 2012 18:18:29 GMT

Creating a New Protocol

←Older revision		Revision as of 18:18, 12 July 2012
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	#Check your work by clicking the http://openwetware.org/images/2/28/ShowPreviewButton.jpg button.		#Check your work by clicking the http://openwetware.org/images/2/28/ShowPreviewButton.jpg button.
	#Save the changes by clicking http://openwetware.org/images/7/71/SavePageButton.jpg ... and you're off.		#Save the changes by clicking http://openwetware.org/images/7/71/SavePageButton.jpg ... and you're off.
		+
		+
		+	==Overview==
		+
		+	Replace this sentence with a brief description of the protocol and its goal.
		+
		+	==Materials==
		+
		+	For a 50 μL PCR reaction:
		+
		+	* 35 μL H<sub>2</sub>O
		+	* 5 μL 10X PCR buffer
		+	* 5 μL 2mM dNTPs (each)
		+	* 1.5 μL 50mM MgCl<sub>2</sub>
		+	* 1 μL 50μM sense primer
		+	* 1 μL 50μM antisense primer
		+	* 1 μL 5nM DNA template
		+	* 0.5 μL TAQ DNA polyermerase
		+
		+	==Procedure==
		+	# In a PCR tube, mix the components on ice in the order they are listed above.
		+	# Perform thermocycling program
		+	## 95 °C 5 min
		+	## 95 °C 30 s
		+	## T<sub>H</sub> 30 s
		+	## 72 °C 1 min for each 1 kb PCR product
		+	## Repeat steps 2-4 a total of 12-36 times (24 is standard).
		+	## 72 °C 5 min
		+	## 12 °C hold
		+
		+	==Notes==
		+	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
		+	#List troubleshooting tips here.
		+	#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
		+	#Anecdotal observations that might be of use to others can also be posted here.
		+
		+	Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
		+
		+	==References==
		+	'''Relevant papers and books'''
		+	<!-- If this protocol has papers or books associated with it, list those references here.-->
		+	<!-- See the [[OpenWetWare:Biblio]] page for more information, but this format doesn't work currently.
		+	<biblio>
		+	#Goldbeter-PNAS-1981 pmid=6947258
		+	#Jacob-JMB-1961 pmid=13718526
		+	#Ptashne-Genetic-Switch isbn=0879697164
		+	</biblio>-->
		+	<!-- Try the [[Template:FormatRef\|FormatRef template]]-->
		+	#{{FormatRef\|Goldbeter, A and Koshland, DE\|1981\| \|Proc Natl Acad Sci U S A 78(11)\|6840-4\| }} PMID 6947258
		+	#{{FormatRef\|Jacob, F and Monod, J J\|1961\| \|Mol Biol 3(3)\|318-56\| }} PMID 13718526
		+	#{{FormatRef\|Ptashne, M\|2004\|Genetic Switch: Phage Lambda Revisited\| \| \|Cold Spring Harbor Laboratory Press}} ISBN 0879697164
		+
		+	==Contact==
		+	*Who has experience with this protocol?
		+
		+	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].
		+
		+	<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. -->
		+
		+	<!-- Move the relevant categories above this line to tag your protocol with the label
		+	[[Category:Protocol]]
		+
		+	[[Category:Needs attention]]
		+
		+	[[Category:In vitro]]
		+
		+	[[Category:In vivo]]
		+
		+	[[Category:In silico]]
		+
		+	[[Category:DNA]]
		+
		+	[[Category:RNA]]
		+
		+	[[Category:Protein]]
		+
		+	[[Category:Chemical]]
		+
		+	[[Category:Escherichia coli]]
		+
		+	[[Category:Yeast]]
		+	-->

Barry Canton at 22:24, 18 May 2007

Barry Canton — Fri, 18 May 2007 22:24:00 GMT

←Older revision		Revision as of 22:24, 18 May 2007
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	#View and copy the source from the [[Protocols/Template\|'''protocols template''']] into your new page and begin editing. '''Make sure you are not editing this page.'''		#View and copy the source from the [[Protocols/Template\|'''protocols template''']] into your new page and begin editing. '''Make sure you are not editing this page.'''
	#Check your work by clicking the http://openwetware.org/images/2/28/ShowPreviewButton.jpg button.		#Check your work by clicking the http://openwetware.org/images/2/28/ShowPreviewButton.jpg button.
-	#Save the changes by clicking ~~[[Image~~:SavePageButton.jpg]] ... and you're off.	+	#Save the changes by clicking http://openwetware.org/images/7/71/SavePageButton.jpg ... and you're off.

Barry Canton at 22:22, 18 May 2007

Barry Canton — Fri, 18 May 2007 22:22:45 GMT

←Older revision		Revision as of 22:22, 18 May 2007
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	#Click on the create this page link.		#Click on the create this page link.
	#View and copy the source from the [[Protocols/Template\|'''protocols template''']] into your new page and begin editing. '''Make sure you are not editing this page.'''		#View and copy the source from the [[Protocols/Template\|'''protocols template''']] into your new page and begin editing. '''Make sure you are not editing this page.'''
-	#Check your work by clicking the ~~[[Image~~:ShowPreviewButton.jpg]] button.	+	#Check your work by clicking the http://openwetware.org/images/2/28/ShowPreviewButton.jpg button.
	#Save the changes by clicking [[Image:SavePageButton.jpg]] ... and you're off.		#Save the changes by clicking [[Image:SavePageButton.jpg]] ... and you're off.

Reshma P. Shetty: made explanation more detailed

Reshma P. Shetty — Fri, 18 May 2007 20:26:04 GMT

made explanation more detailed

←Older revision		Revision as of 20:26, 18 May 2007
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	==Creating a New Protocol==		==Creating a New Protocol==
-	* [[~~OpenWetWare~~:~~Searching~~ \| ~~Search~~]] ~~for your protocol or~~ page ~~to verify that~~ is ~~not~~ already ~~in existence~~	+	#In the search box on the lefthand side navigation bar, type <code>Protocol name</code>. If it is a protocol specific to your lab, type <code>LabName:Protocol name</code> (be sure and replace <code>LabName</code> with your PI's last name like <code>Knight</code> or <code>Smolke</code> [[Image:Searchbox.png\|right]]
-	* Click on "create this page"	+	#You should see a page come up with a message saying '''"There is no page titled "Protocol name".''' You can create this page." If you don't see this message then that means a page with that name already exists. You'll need to choose a different protocol name. [[Image:SearchforProtocol.tiff\|450px\|left]] <br style="clear:both" />
-	* View and copy the source from the [[Protocols/Template\|'''protocols template''']] ~~in to~~ your new page and begin editing. '''Make sure you are not editing this page.'''	+	#Click on the create this page link.
		+	#View and copy the source from the [[Protocols/Template\|'''protocols template''']] into your new page and begin editing. '''Make sure you are not editing this page.'''
		+	#Check your work by clicking the [[Image:ShowPreviewButton.jpg]] button.
		+	#Save the changes by clicking [[Image:SavePageButton.jpg]] ... and you're off.

Austin J. Che at 12:56, 18 May 2007

Austin J. Che — Fri, 18 May 2007 12:56:13 GMT

←Older revision		Revision as of 12:56, 18 May 2007
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	* [[OpenWetWare:Searching \| Search]] for your protocol or page to verify that is not already in existence		* [[OpenWetWare:Searching \| Search]] for your protocol or page to verify that is not already in existence
	* Click on "create this page"		* Click on "create this page"
-	* View and copy the source from the [[Protocols/Template\|'''protocols template''']] in to your new page and begin editing.	+	* View and copy the source from the [[Protocols/Template\|'''protocols template''']] in to your new page and begin editing. '''Make sure you are not editing this page.'''

Austin J. Che at 12:55, 18 May 2007

Austin J. Che — Fri, 18 May 2007 12:55:13 GMT

←Older revision		Revision as of 12:55, 18 May 2007
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-	~~#REDIRECT~~ [[~~Marek~~: ~~Freeze-down~~/~~Thaw~~]]	+	==Creating a New Protocol==
		+	* [[OpenWetWare:Searching \| Search]] for your protocol or page to verify that is not already in existence
		+	* Click on "create this page"
		+	* View and copy the source from the [[Protocols/Template\|'''protocols template''']] in to your new page and begin editing.