DNA ligation - Revision history

Christopher C Vanlang: /* Specific protocols */

2012-07-01T10:41:03Z

Specific protocols

←Older revision		Revision as of 10:41, 1 July 2012
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	==Specific protocols==		==Specific protocols==
-	[[Endy:DNA ligation using T4 DNA ligase]] -- Using T4 DNA Ligase	+	*[[Endy:DNA ligation using T4 DNA ligase]] -- Using T4 DNA Ligase
		+	*[[Knight:DNA ligation using NEB Quick Ligation Kit]] -- 5min ligation.
		+	*[[Knight:TOPO TA cloning]] -- For PCR products.
		+	*[[Silver: Ligation\|Silver:Ligation]] -- A protocol for sticky end ligations using the Roche Kit.
		+	*[[Richard_Lab:Ligation]] -- Uses T4 Ligase
		+	*[[BE.109:DNA engineering/DNA ligation and bacterial transformation\|BE.109:DNA ligation]] -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks.

-	[[~~Knight~~:~~DNA ligation using NEB Quick Ligation Kit~~]] ~~-- 5min ligation.~~	+	*[[Corum:T4_Ligation]]
-		+
-	~~[[Knight:TOPO TA cloning]] -- For PCR products.~~	+
-		+
-	~~[[Silver: Ligation\|Silver:Ligation]] -- A protocol for sticky end ligations using the Roche Kit.~~	+
-		+
-	~~[[Richard_Lab:Ligation]] -- Uses T4 Ligase~~	+
-		+
-	[[BE.109:DNA engineering/DNA ligation and bacterial transformation\|BE.109:DNA ligation]] -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks.	+

	==Discussion==		==Discussion==

Christopher C Vanlang: /* Notes */

2012-01-02T03:10:42Z

Notes

←Older revision		Revision as of 03:10, 2 January 2012
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	#Using [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA Gel Stain] is a safer, non-carcinogenic alternative to ethidium bromide.		#Using [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA Gel Stain] is a safer, non-carcinogenic alternative to ethidium bromide.
	#T4 DNA Ligase is very sensitive to shear, so spinning your ligation mix or vortexing it to mix it can affect your yields. Instead try mixing with the pipette tip or slowly resuspending the solution.		#T4 DNA Ligase is very sensitive to shear, so spinning your ligation mix or vortexing it to mix it can affect your yields. Instead try mixing with the pipette tip or slowly resuspending the solution.
		+	#If there is a lot of self-ligation look into [[Phosphatase treatment of linearized vector]].

	==Acknowledgments==		==Acknowledgments==

Michael A. Speer: /* Specific protocols */

2011-01-25T22:27:16Z

Specific protocols

←Older revision		Revision as of 22:27, 25 January 2011
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	[[Silver: Ligation\|Silver:Ligation]] -- A protocol for sticky end ligations using the Roche Kit.		[[Silver: Ligation\|Silver:Ligation]] -- A protocol for sticky end ligations using the Roche Kit.

-	[[Richard_Lab:Ligation]]	+	[[Richard_Lab:Ligation]] -- Uses T4 Ligase

	[[BE.109:DNA engineering/DNA ligation and bacterial transformation\|BE.109:DNA ligation]] -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks.		[[BE.109:DNA engineering/DNA ligation and bacterial transformation\|BE.109:DNA ligation]] -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks.

Tim Hsiau at 00:53, 19 January 2010

2010-01-19T00:53:06Z

←Older revision		Revision as of 00:53, 19 January 2010
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	# Crowe-NAR-1991 pmid=2011503		# Crowe-NAR-1991 pmid=2011503
	# Olivera-PNAS-1967 pmid=5341238		# Olivera-PNAS-1967 pmid=5341238
-	~~# Michelsen-Anal-1995 pmid=7778774~~
	// DNA ligation by ''Escherichia coli'' DNA ligase		// DNA ligation by ''Escherichia coli'' DNA ligase
		+	# Michelsen-Anal-1995 pmid=7778774
		+
	</biblio>		</biblio>

Tim Hsiau at 23:10, 17 January 2010

2010-01-17T23:10:39Z

←Older revision		Revision as of 23:10, 17 January 2010
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	#If you are having trouble with your ligation, NEB offers FAQ's ([http://www.neb.com/nebecomm/products/faqproductM2200.asp Quick Ligation] [http://www.neb.com/nebecomm/products/faqproductM0202.asp T4 DNA ligase]) and [http://www.neb.com/nebecomm/tech_reference/dna_rna/tips.asp tips] to help.		#If you are having trouble with your ligation, NEB offers FAQ's ([http://www.neb.com/nebecomm/products/faqproductM2200.asp Quick Ligation] [http://www.neb.com/nebecomm/products/faqproductM0202.asp T4 DNA ligase]) and [http://www.neb.com/nebecomm/tech_reference/dna_rna/tips.asp tips] to help.
	#Prior to the ligation, some heat their DNA slightly (maybe ~37°C) to melt any sticky ends which may have annealed improperly at low temperatures.		#Prior to the ligation, some heat their DNA slightly (maybe ~37°C) to melt any sticky ends which may have annealed improperly at low temperatures.
-	#[[Tom Knight]] has read that ligase can inhibit transformation. By heat-inactivating the ligase, this inhibition can be avoided. However, according to the NEB FAQ, heat-inactivation of PEG (which is present in the ligation reaction) also inhibits transformation, therefore a spin-column purification is recommended prior to transformation if you are having problems.	+	#[[Tom Knight]] has read that ligase can inhibit transformation <cite>Michelsen-Anal-1995</cite>. By heat-inactivating the ligase, this inhibition can be avoided. However, according to the NEB FAQ, heat-inactivation of PEG (which is present in the ligation reaction) also inhibits transformation, therefore a spin-column purification is recommended prior to transformation if you are having problems.
	#Treating PCR products with proteinase K prior to restriction digest dramatically improves the efficiency of subsequent ligation reactions. <cite>Crowe-NAR-1991</cite>		#Treating PCR products with proteinase K prior to restriction digest dramatically improves the efficiency of subsequent ligation reactions. <cite>Crowe-NAR-1991</cite>
	#Using [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA Gel Stain] is a safer, non-carcinogenic alternative to ethidium bromide.		#Using [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA Gel Stain] is a safer, non-carcinogenic alternative to ethidium bromide.

Tim Hsiau: /* References */

2010-01-17T23:10:05Z

References

←Older revision		Revision as of 23:10, 17 January 2010
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	# Crowe-NAR-1991 pmid=2011503		# Crowe-NAR-1991 pmid=2011503
	# Olivera-PNAS-1967 pmid=5341238		# Olivera-PNAS-1967 pmid=5341238
		+	# Michelsen-Anal-1995 pmid=7778774
	// DNA ligation by ''Escherichia coli'' DNA ligase		// DNA ligation by ''Escherichia coli'' DNA ligase
	</biblio>		</biblio>

Michael A. Speer: /* Specific protocols */

2009-06-18T18:59:14Z

Specific protocols

←Older revision		Revision as of 18:59, 18 June 2009
Line 98:		Line 98:

	[[Silver: Ligation\|Silver:Ligation]] -- A protocol for sticky end ligations using the Roche Kit.		[[Silver: Ligation\|Silver:Ligation]] -- A protocol for sticky end ligations using the Roche Kit.
		+
		+	[[Richard_Lab:Ligation]]

	[[BE.109:DNA engineering/DNA ligation and bacterial transformation\|BE.109:DNA ligation]] -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks.		[[BE.109:DNA engineering/DNA ligation and bacterial transformation\|BE.109:DNA ligation]] -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks.

Gan Hm: /* Method */

2009-05-25T04:52:36Z

Method

←Older revision		Revision as of 04:52, 25 May 2009
Line 45:		Line 45:
	#Use disks shiny side up		#Use disks shiny side up
	#Store at -20°C		#Store at -20°C
-
-
-	# Dialyzing the ligation reaction isn't neccesary for electroporation. I have been using the reaction straight for electroporation. Minimize the volume use 1ul for 40-50ul cells . Arching shouldn't occur at all.

	==Critical steps==		==Critical steps==

Gan Hm: /* Method */

2009-05-25T04:51:45Z

Method

←Older revision		Revision as of 04:51, 25 May 2009
Line 45:		Line 45:
	#Use disks shiny side up		#Use disks shiny side up
	#Store at -20°C		#Store at -20°C
		+
		+
		+	# Dialyzing the ligation reaction isn't neccesary for electroporation. I have been using the reaction straight for electroporation. Minimize the volume use 1ul for 40-50ul cells . Arching shouldn't occur at all.

	==Critical steps==		==Critical steps==

Ellis: /* Factors affecting efficiency */

2009-04-29T10:21:14Z

Factors affecting efficiency

←Older revision		Revision as of 10:21, 29 April 2009
Line 65:		Line 65:

	Ligation efficiency was '''severely decreased''' (x10000) by		Ligation efficiency was '''severely decreased''' (x10000) by
-	#Using DNA fragments that have been exposed to ~~ethidium bromide and~~ UV during the gel extraction procedure (''can avoid by blind excision, or by using a black-light or 365nm UV transilluminator'')	+	#Using DNA fragments that have been exposed to UV during the gel extraction procedure (''can avoid by blind excision, or by using a black-light or 365nm UV transilluminator instead of the usual 312nm type'')
	#Using the NEB Quick Ligation Kit (''heat inactivation of PEG in the buffer ruins transformation, without heat inactivation the ligation probably would've been fine'')		#Using the NEB Quick Ligation Kit (''heat inactivation of PEG in the buffer ruins transformation, without heat inactivation the ligation probably would've been fine'')