Corum:T4 Ligation - Revision history

Sean P Corum: /* Materials */

Sean P Corum — Thu, 30 Aug 2012 22:05:03 GMT

Materials

←Older revision		Revision as of 22:05, 30 August 2012
Line 9:		Line 9:
	For a 10 μL ligation reaction:		For a 10 μL ligation reaction:

		+	* 3 μL sterile ddH<sub>2</sub>O
		+	* 1 μL 10X ligation buffer
	* 5 μL ~100nM linker dsDNA (either hybridization product or digested, purified PCR product)		* 5 μL ~100nM linker dsDNA (either hybridization product or digested, purified PCR product)
	* 0.5 μL ~10nM backbone dsDNA (gel extracted digestion product from donor plasmid)		* 0.5 μL ~10nM backbone dsDNA (gel extracted digestion product from donor plasmid)
-	* 2 μL sterile ddH<sub>2</sub>O
-	* 1 μL 10X ligation buffer
-	* 1 μL 10mg/ml BSA
	* 0.5 μL T4 ligase		* 0.5 μL T4 ligase
-	(Note: ~~standard~~ ratio of backbone to linker given here is ~1:100. This may need to be adjusted ~~for non-standard conditions~~.)	+	(Note: ratio of backbone to linker given here is ~1:100. This may need to be adjusted in some cases.)

	==Procedure==		==Procedure==

Sean P Corum: /* Notes */

Sean P Corum — Thu, 30 Aug 2012 22:03:50 GMT

Notes

←Older revision		Revision as of 22:03, 30 August 2012
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	Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
		+
		+	-------------
		+	# *'''SC 18:03, 30 August 2012 (EDT)''': Removed 1mg/ml BSA component from protocol, per the fact that it is not recommended in the [http://www.neb.com/nebecomm/products/productm0202.asp NEB protocol].

	==References==		==References==

Sean P Corum: /* Materials */

Sean P Corum — Thu, 26 Jul 2012 17:38:19 GMT

Materials

←Older revision		Revision as of 17:38, 26 July 2012
Line 9:		Line 9:
	For a 10 μL ligation reaction:		For a 10 μL ligation reaction:

-	* 5 μL linker dsDNA (either ~~100nM standard concentration linker~~ hybridization product or ~~standard~~ digested ~~and~~ purified PCR product~~, which is usually ~100nM~~)	+	* 5 μL ~100nM linker dsDNA (either hybridization product or digested, purified PCR product)
-	* 0.5 μL backbone dsDNA (~~usually ~10 nM) (Note: standard ratio of backbone to linker is ~1:100. This may need to be adjusted for non-standard conditions.~~)	+	* 0.5 μL ~10nM backbone dsDNA (gel extracted digestion product from donor plasmid)
	* 2 μL sterile ddH<sub>2</sub>O		* 2 μL sterile ddH<sub>2</sub>O
	* 1 μL 10X ligation buffer		* 1 μL 10X ligation buffer
	* 1 μL 10mg/ml BSA		* 1 μL 10mg/ml BSA
	* 0.5 μL T4 ligase		* 0.5 μL T4 ligase
		+	(Note: standard ratio of backbone to linker given here is ~1:100. This may need to be adjusted for non-standard conditions.)

	==Procedure==		==Procedure==

Sean P Corum: /* Procedure */

Sean P Corum — Thu, 26 Jul 2012 17:34:39 GMT

Procedure

←Older revision		Revision as of 17:34, 26 July 2012
Line 19:		Line 19:

	# Add the reaction components together in order listed above. Mix gently and spin.		# Add the reaction components together in order listed above. Mix gently and spin.
		+	# Include -linker control reaction (sub sterile ddH<sub>2</sub>O for linker).
	# Incubate 16 °C overnight (8 hr minimum).		# Incubate 16 °C overnight (8 hr minimum).
	# [http://openwetware.org/wiki/Corum:Transformation Transform] 2.5 μL rea(or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.		# [http://openwetware.org/wiki/Corum:Transformation Transform] 2.5 μL rea(or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.

Sean P Corum: /* Procedure */

Sean P Corum — Thu, 26 Jul 2012 17:33:36 GMT

Procedure

←Older revision		Revision as of 17:33, 26 July 2012
Line 20:		Line 20:
	# Add the reaction components together in order listed above. Mix gently and spin.		# Add the reaction components together in order listed above. Mix gently and spin.
	# Incubate 16 °C overnight (8 hr minimum).		# Incubate 16 °C overnight (8 hr minimum).
-	# [http://openwetware.org/wiki/Corum:Transformation Transform] 2.5 μL (or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.	+	# [http://openwetware.org/wiki/Corum:Transformation Transform] 2.5 μL rea(or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.

	==Notes==		==Notes==

Sean P Corum: /* References */

Sean P Corum — Thu, 26 Jul 2012 17:32:15 GMT

References

←Older revision		Revision as of 17:32, 26 July 2012
Line 31:		Line 31:

	==References==		==References==
-	~~'''Relevant papers, books, and websites'''~~
-	~~<!-- If this protocol has papers or books associated with it, list those references here.-->~~
-	~~<!-- Try the [[Template:FormatRef\|FormatRef template]]-->~~
	#[http://www.neb.com New England Biolabs]		#[http://www.neb.com New England Biolabs]

Sean P Corum: /* Procedure */

Sean P Corum — Thu, 26 Jul 2012 17:30:56 GMT

Procedure

←Older revision		Revision as of 17:30, 26 July 2012
Line 20:		Line 20:
	# Add the reaction components together in order listed above. Mix gently and spin.		# Add the reaction components together in order listed above. Mix gently and spin.
	# Incubate 16 °C overnight (8 hr minimum).		# Incubate 16 °C overnight (8 hr minimum).
-	# [http://openwetware.org/wiki/Corum:Transformation Transform] 2.5 μL (or more, if necessary, to obtain an acceptable amount of colonies~~, for example~~) into chemically competent cells.	+	# [http://openwetware.org/wiki/Corum:Transformation Transform] 2.5 μL (or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.

	==Notes==		==Notes==

Sean P Corum: /* Procedure */

Sean P Corum — Thu, 26 Jul 2012 17:30:33 GMT

Procedure

←Older revision		Revision as of 17:30, 26 July 2012
Line 20:		Line 20:
	# Add the reaction components together in order listed above. Mix gently and spin.		# Add the reaction components together in order listed above. Mix gently and spin.
	# Incubate 16 °C overnight (8 hr minimum).		# Incubate 16 °C overnight (8 hr minimum).
		+	# [http://openwetware.org/wiki/Corum:Transformation Transform] 2.5 μL (or more, if necessary, to obtain an acceptable amount of colonies, for example) into chemically competent cells.

	==Notes==		==Notes==

Sean P Corum: /* Materials */

Sean P Corum — Thu, 26 Jul 2012 17:28:35 GMT

Materials

←Older revision		Revision as of 17:28, 26 July 2012
Line 10:		Line 10:

	* 5 μL linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)		* 5 μL linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)
-	* 0.5 μL backbone dsDNA (usually ~10 nM) (Note: ratio of backbone to linker ~~should be~~ 1:100. ~~The specific amounts of linker and backbone dsDNAs~~ may need to be adjusted ~~to keep this ratio~~ for non-standard ~~concentrations~~)	+	* 0.5 μL backbone dsDNA (usually ~10 nM) (Note: standard ratio of backbone to linker is ~1:100. This may need to be adjusted for non-standard conditions.)
	* 2 μL sterile ddH<sub>2</sub>O		* 2 μL sterile ddH<sub>2</sub>O
	* 1 μL 10X ligation buffer		* 1 μL 10X ligation buffer

Sean P Corum at 15:35, 29 June 2012

Sean P Corum — Fri, 29 Jun 2012 15:35:22 GMT

←Older revision		Revision as of 15:35, 29 June 2012
Line 9:		Line 9:
	For a 10 μL ligation reaction:		For a 10 μL ligation reaction:

-	* 7 μL linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)	+	* 5 μL linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)
-	* 0.5 μL backbone dsDNA (usually ~10 nM) (Note: ratio of backbone to linker should be ~~10-20~~:1. The specific amounts of linker and backbone dsDNAs may need to be adjusted to keep this ratio for non-standard concentrations)	+	* 0.5 μL backbone dsDNA (usually ~10 nM) (Note: ratio of backbone to linker should be 1:100. The specific amounts of linker and backbone dsDNAs may need to be adjusted to keep this ratio for non-standard concentrations)
		+	* 2 μL sterile ddH<sub>2</sub>O
	* 1 μL 10X ligation buffer		* 1 μL 10X ligation buffer
	* 1 μL 10mg/ml BSA		* 1 μL 10mg/ml BSA