Richard Lab:Ligation - Revision history

Michael A. Speer: /* Procedure */

Michael A. Speer — Tue, 29 Mar 2011 19:13:59 GMT

Procedure

←Older revision		Revision as of 19:13, 29 March 2011
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	::a. 11µl of deionized water		::a. 11µl of deionized water
	::b. 2µl of Reaction Buffer for T4 Ligase.		::b. 2µl of Reaction Buffer for T4 Ligase.
-	::c. ~~3µl~~ digested "Vector" (Phosphatased).	+	::c. 2µl digested "Vector" (Phosphatased).
-	::d. ~~3µl~~ digested "Insert" (PCR product digested with DpnI).	+	::d. 4µl digested "Insert" (PCR product digested with DpnI).
	::e. 1µl of [[T4_DNA_ligase\|DNA Ligase]].		::e. 1µl of [[T4_DNA_ligase\|DNA Ligase]].
	2. Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br>		2. Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br>

Michael A. Speer at 19:12, 29 March 2011

Michael A. Speer — Tue, 29 Mar 2011 19:12:15 GMT

←Older revision		Revision as of 19:12, 29 March 2011
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	Back to [[Richard_Lab:protocols \| Protocols]]		Back to [[Richard_Lab:protocols \| Protocols]]
	<center>		<center>
-	'''This protocol is for the typical ligation of DNA done during [[Richard Lab:Amplified ~~Insert Assembly~~\|Amplified Insert Assembly]].	+	'''This protocol is for the typical ligation of DNA done during [[Richard Lab:Amplified insert assembly\|Amplified Insert Assembly]].
	'''The [[DNA_ligation \| consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>		'''The [[DNA_ligation \| consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>

Michael A. Speer at 19:09, 29 March 2011

Michael A. Speer — Tue, 29 Mar 2011 19:09:40 GMT

←Older revision		Revision as of 19:09, 29 March 2011
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	Back to [[Richard_Lab:protocols \| Protocols]]		Back to [[Richard_Lab:protocols \| Protocols]]
	<center>		<center>
-	'''This protocol is for the typical ligation of DNA done ~~using the~~ [[Richard Lab:~~Bio-Brick~~ Assembly ~~Schedule~~\|~~Richard Lab New Standard~~ Assembly]].	+	'''This protocol is for the typical ligation of DNA done during [[Richard Lab:Amplified Insert Assembly\|Amplified Insert Assembly]].
	'''The [[DNA_ligation \| consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>		'''The [[DNA_ligation \| consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>

Michael A. Speer at 22:28, 25 January 2011

Michael A. Speer — Tue, 25 Jan 2011 22:28:44 GMT

←Older revision		Revision as of 22:28, 25 January 2011
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	Back to [[Richard_Lab:protocols \| Protocols]]		Back to [[Richard_Lab:protocols \| Protocols]]
	<center>		<center>
-	'''This protocol is for the typical ligation of DNA done using the [[Richard Lab:Bio-Brick Assembly Schedule\|Richard Lab Standard Assembly ~~2.0~~]].	+	'''This protocol is for the typical ligation of DNA done using the [[Richard Lab:Bio-Brick Assembly Schedule\|Richard Lab New Standard Assembly]].
	'''The [[DNA_ligation \| consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>		'''The [[DNA_ligation \| consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>

Michael A. Speer at 22:25, 25 January 2011

Michael A. Speer — Tue, 25 Jan 2011 22:25:42 GMT

←Older revision		Revision as of 22:25, 25 January 2011
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		+	Back to [[Richard_Lab:protocols \| Protocols]]
	<center>		<center>
	'''This protocol is for the typical ligation of DNA done using the [[Richard Lab:Bio-Brick Assembly Schedule\|Richard Lab Standard Assembly 2.0]].		'''This protocol is for the typical ligation of DNA done using the [[Richard Lab:Bio-Brick Assembly Schedule\|Richard Lab Standard Assembly 2.0]].
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	===Notes===		===Notes===
	* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.		* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.
		+
		+	Back to [[Richard_Lab:protocols \| Protocols]]

Michael A. Speer: /* Notes */

Michael A. Speer — Tue, 25 Jan 2011 22:24:41 GMT

Notes

←Older revision		Revision as of 22:24, 25 January 2011
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	===Notes===		===Notes===
-	* [[User:Michael A. Speer\|Mike]] has had problems with blunt end ligation if this reaction is let go for too long.
	* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.		* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.

Michael A. Speer: /* Procedure */

Michael A. Speer — Tue, 25 Jan 2011 22:23:55 GMT

Procedure

←Older revision		Revision as of 22:23, 25 January 2011
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	::e. 1µl of [[T4_DNA_ligase\|DNA Ligase]].		::e. 1µl of [[T4_DNA_ligase\|DNA Ligase]].
	2. Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br>		2. Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br>
-	3. Kill Reaction for 15 mins at 65°C	+	3. Kill Reaction for 15 mins at 65°C '''Do not forget this step'''

	===Notes===		===Notes===
	* [[User:Michael A. Speer\|Mike]] has had problems with blunt end ligation if this reaction is let go for too long.		* [[User:Michael A. Speer\|Mike]] has had problems with blunt end ligation if this reaction is let go for too long.
	* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.		* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.

Michael A. Speer: /* Procedure */

Michael A. Speer — Wed, 12 Jan 2011 23:50:43 GMT

Procedure

←Older revision		Revision as of 23:50, 12 January 2011
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	::b. 2µl of Reaction Buffer for T4 Ligase.		::b. 2µl of Reaction Buffer for T4 Ligase.
	::c. 3µl digested "Vector" (Phosphatased).		::c. 3µl digested "Vector" (Phosphatased).
-	::d. 3µl digested "Insert" (PCR product with DpnI).	+	::d. 3µl digested "Insert" (PCR product digested with DpnI).
	::e. 1µl of [[T4_DNA_ligase\|DNA Ligase]].		::e. 1µl of [[T4_DNA_ligase\|DNA Ligase]].
	2. Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br>		2. Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br>

Michael A. Speer at 23:49, 12 January 2011

Michael A. Speer — Wed, 12 Jan 2011 23:49:35 GMT

←Older revision		Revision as of 23:49, 12 January 2011
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	<center>		<center>
-	'''This protocol is for the typical ligation of DNA done in the Richard Lab.	+	'''This protocol is for the typical ligation of DNA done using the [[Richard Lab:Bio-Brick Assembly Schedule\|Richard Lab Standard Assembly 2.0]].
	'''The [[DNA_ligation \| consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>		'''The [[DNA_ligation \| consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>

Michael A. Speer: /* Procedure */

Michael A. Speer — Wed, 12 Jan 2011 23:46:21 GMT

Procedure

←Older revision		Revision as of 23:46, 12 January 2011
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	1.Mix together the following:		1.Mix together the following:
-	::a. ~~5µl digested front part (EcoRI and SpeI).~~	+	::a. 11µl of deionized water
-	::b. ~~5µl digested rear part (XbaI and PstI)~~.	+	::b. 2µl of Reaction Buffer for T4 Ligase.
-	::c. ~~4µl deionized water~~.	+	::c. 3µl digested "Vector" (Phosphatased).
-	::d. 3µl digested ~~vector~~ (~~EcoRI and PstI~~).	+	::d. 3µl digested "Insert" (PCR product with DpnI).
-	::e. ~~2µl of Reaction Buffer for T4 Ligase.~~	+	::e. 1µl of [[T4_DNA_ligase\|DNA Ligase]].
-	~~::f.~~ 1µl of [[T4_DNA_ligase\|DNA Ligase]].	+	2. Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br>
-	2. Incubate at room-temp for 10 mins (or at 4°C overnight)<br>	+	3. Kill Reaction for 15 mins at 65°C
-	3. Kill Reaction for 15 mins at ~~80°C~~	+

	===Notes===		===Notes===
	* [[User:Michael A. Speer\|Mike]] has had problems with blunt end ligation if this reaction is let go for too long.		* [[User:Michael A. Speer\|Mike]] has had problems with blunt end ligation if this reaction is let go for too long.
	* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.		* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.