Corum:digest - Revision history

Sean P Corum at 18:53, 19 June 2012

2012-06-19T18:53:07Z

←Older revision		Revision as of 18:53, 19 June 2012
Line 1:		Line 1:
-	*'''SC 14:47, 19 June 2012 (EDT)''':	+	'''SC 14:53, 19 June 2012 (EDT)''':

	==Overview==		==Overview==
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	[[Category:In vitro]]		[[Category:In vitro]]

-	[[Category:~~Digestions~~]]	+	[[Category:Digestion]]
		+
		+	[[Category:Restriction Enzyme]]

Sean P Corum at 18:50, 19 June 2012

2012-06-19T18:50:31Z

←Older revision		Revision as of 18:50, 19 June 2012
Line 2:		Line 2:

	==Overview==		==Overview==
-	Standard DNA digest protocol using [http://www.neb.com \| NEB] restriction enzymes.	+	Standard DNA digest protocol using [http://www.neb.com NEB] restriction enzymes.

	==Materials==		==Materials==
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	* ~0.8V DNA		* ~0.8V DNA
-	* 0.1V 10X digestion buffer (1, 2, 3, or 4; NEB)	+	* 0.1V 10X digestion buffer (1, 2, 3, or 4; [http://www.neb.com NEB])
	* 0.1V 10mg/ml BSA		* 0.1V 10mg/ml BSA
	* 1 μL restriction enzyme 1		* 1 μL restriction enzyme 1
Line 15:		Line 15:

	==Procedure==		==Procedure==
-	# Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com \| NEB].	+	# Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com NEB].
	# Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.		# Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.
	# Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.		# Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.
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	==References==		==References==
-	'''Relevant papers ~~and~~ books'''	+	'''Relevant papers, books, and websites'''
	<!-- If this protocol has papers or books associated with it, list those references here.-->		<!-- If this protocol has papers or books associated with it, list those references here.-->
	<!-- Try the [[Template:FormatRef\|FormatRef template]]-->		<!-- Try the [[Template:FormatRef\|FormatRef template]]-->
-	#[http://www.neb.com \| New England Biolabs]	+	#[http://www.neb.com New England Biolabs]

	==Contact==		==Contact==

Sean P Corum at 18:49, 19 June 2012

2012-06-19T18:49:04Z

←Older revision		Revision as of 18:49, 19 June 2012
Line 2:		Line 2:

	==Overview==		==Overview==
-	Standard DNA digest protocol (NEB restriction enzymes).	+	Standard DNA digest protocol using [http://www.neb.com \| NEB] restriction enzymes.

	==Materials==		==Materials==
Line 15:		Line 15:

	==Procedure==		==Procedure==
-	# Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com\|NEB].	+	# Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com \| NEB].
	# Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.		# Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.
	# Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.		# Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.
Line 33:		Line 33:
	<!-- If this protocol has papers or books associated with it, list those references here.-->		<!-- If this protocol has papers or books associated with it, list those references here.-->
	<!-- Try the [[Template:FormatRef\|FormatRef template]]-->		<!-- Try the [[Template:FormatRef\|FormatRef template]]-->
-	#~~{{FormatRef\|Sambrook, J and Russell, DW\|2001\|Molecular Cloning~~: ~~A Laboratory Manual (Volume II)\| \|~~ \|~~Cold Spring Harbor Laboratory Press}} ISBN 0879695773~~	+	#[http://www.neb.com \| New England Biolabs]

	==Contact==		==Contact==

Sean P Corum at 18:47, 19 June 2012

2012-06-19T18:47:02Z

←Older revision		Revision as of 18:47, 19 June 2012
Line 1:		Line 1:
		+	*'''SC 14:47, 19 June 2012 (EDT)''':
		+
	==Overview==		==Overview==
-	Standard ~~polymerase chain reaction~~ (~~PCR~~) ~~protocol~~.	+	Standard DNA digest protocol (NEB restriction enzymes).

	==Materials==		==Materials==

-	For a 50 μL ~~PCR reaction~~:	+	For a DNA digestion of total volume V (in μL):

-	* ~~35 μL H<sub>2</sub>O~~	+	* ~0.8V DNA
-	* ~~5 μL~~ 10X ~~PCR~~ buffer	+	* 0.1V 10X digestion buffer (1, 2, 3, or 4; NEB)
-	* 5 μL 2mM dNTPs (~~each~~)	+	* 0.1V 10mg/ml BSA
-	* 1.~~5 μL 50mM MgCl<sub>2<~~/~~sub>~~	+	* 1 μL restriction enzyme 1
-	* 1 μL ~~50μM sense primer~~	+	* 1 μL restriction enzyme 2 (optional, for double digestion)
-	* 1 ~~μL 50μM antisense primer~~	+
-	* 1 μL ~~5nM DNA template~~	+
-	* 0.5 μL TAQ DNA polyermerase	+

	==Procedure==		==Procedure==
-	# ~~In a PCR tube~~, ~~mix the components on ice in the order they are listed above.~~	+	# Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com\|NEB].
-	~~# Perform the following thermocycling program~~:	+	# Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.
-	~~## 95 °C 5 min~~	+	# Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.
-	~~## 95 °C 30 s~~	+	# Add the reaction components together in order listed above. Mix gently and spin.
-	~~## T<sub>H<~~/~~sub> 30 s~~	+	# Incubate at reaction temperature for 3 hr to overnight.
-	#~~# 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==		==Notes==
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	==Contact==		==Contact==
	* [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum]		* [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum]
-	* '''SC 17:44, 18 June 2012 (EDT)''':

	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].		or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].
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	[[Category:In vitro]]		[[Category:In vitro]]

-	[[Category:~~PCR~~]]	+	[[Category:Digestions]]

Sean P Corum: New page: ==Overview== Standard polymerase chain reaction (PCR) protocol. ==Materials== For a 50 μL PCR reaction: * 35 μL H₂O * 5 μL 10X PCR buffer * 5 μL 2mM dNTPs (each) * 1.5 μ...

2012-06-19T18:31:54Z

New page: ==Overview== Standard polymerase chain reaction (PCR) protocol. ==Materials== For a 50 μL PCR reaction: * 35 μL H2O * 5 μL 10X PCR buffer * 5 μL 2mM dNTPs (each) * 1.5 μ...

New page

==Overview==
Standard polymerase chain reaction (PCR) protocol.

==Materials==

For a 50 μL PCR reaction:

* 35 μL H2O
* 5 μL 10X PCR buffer
* 5 μL 2mM dNTPs (each)
* 1.5 μL 50mM MgCl2
* 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 the following thermocycling program:
## 95 °C 5 min
## 95 °C 30 s
## TH 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 <nowiki>'''*~~~~''':</nowiki> to the beginning of your tip.

==References==
'''Relevant papers and books'''


#{{FormatRef|Sambrook, J and Russell, DW|2001|Molecular Cloning: A Laboratory Manual (Volume II)| | |Cold Spring Harbor Laboratory Press}} ISBN 0879695773

==Contact==
* [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum]
* '''SC 17:44, 18 June 2012 (EDT)''':

or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].



[[Category:Protocol]]

[[Category:DNA]]

[[Category:In vitro]]

[[Category:PCR]]

Corum:digest - Revision history

Sean P Corum at 18:53, 19 June 2012

Sean P Corum at 18:50, 19 June 2012

Sean P Corum at 18:49, 19 June 2012

Sean P Corum at 18:47, 19 June 2012

Sean P Corum: New page: ==Overview== Standard polymerase chain reaction (PCR) protocol. ==Materials== For a 50 μL PCR reaction: * 35 μL H2O * 5 μL 10X PCR buffer * 5 μL 2mM dNTPs (each) * 1.5 μ...

Sean P Corum: New page: ==Overview== Standard polymerase chain reaction (PCR) protocol. ==Materials== For a 50 μL PCR reaction: * 35 μL H₂O * 5 μL 10X PCR buffer * 5 μL 2mM dNTPs (each) * 1.5 μ...