Restriction Enzyme Based Cloning - Revision history

Davebrid at 14:40, 7 May 2009

2009-05-07T14:40:14Z

← Older revision		Revision as of 14:40, 7 May 2009
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	#Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C. Add 1 uL of CIAP to the vector for the last ~10min at 37C.		#Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C. Add 1 uL of CIAP to the vector for the last ~10min at 37C.
	#Gel purify both vector and insert (Qiagen kit).		#Gel purify both vector and insert (Qiagen kit).
	:*Run out on gel (see [[Preparing an Agarose Gel]]		:*Run out on gel (see [[Preparing an Agarose Gel]])
	:*On a gel box cut out appropriate bands and place in clean eppendorf tubes. Try to limit the exposure to UV light		:*On a gel box cut out appropriate bands and place in clean eppendorf tubes. Try to limit the exposure to UV light
	:*Weigh out gel piece. For each mg add 3 uL of QG (orange) buffer. For example, for a 0.1g piece add 300 uL buffer.		:*Weigh out gel piece. For each mg add 3 uL of QG (orange) buffer. For example, for a 0.1g piece add 300 uL buffer.

Davebrid: updated with gel purification

2009-05-07T14:22:10Z

updated with gel purification

← Older revision		Revision as of 14:22, 7 May 2009
Line 1:		Line 1:
	#If insert is PCR, run PCR then PCR purify (Qiagen kit). Elute in 30 uL EB		#If insert is PCR, run PCR then PCR purify (Qiagen kit). Elute in 30 uL EB
	#Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C. Add 1 uL of CIAP to the vector for the last ~10min at 37C.		#Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C. Add 1 uL of CIAP to the vector for the last ~10min at 37C.
	#Gel purify both vector and insert (Qiagen kit). ~~Elute~~ in 30 uL		#Gel purify both vector and insert (Qiagen kit).
	#Combine 3-6 uL insert with 1-2 uL ~~vector. You want about a 3x excess of insert to~~ vector. Place at 50-65C for 5-10 min to help sticky end binding~~. Also do a no insert negative control~~.		:*Run out on gel (see [[Preparing an Agarose Gel]]
	#Add 2 uL ligase buffer (single use aliquots) ~~and water/EB to 10 uL final volume~~. Add 1 uL T4 DNA Ligase (Invitrogen).		:*On a gel box cut out appropriate bands and place in clean eppendorf tubes. Try to limit the exposure to UV light
	#Incubate 1h-O/N at 16C (water bath in cold room).		:*Weigh out gel piece. For each mg add 3 uL of QG (orange) buffer. For example, for a 0.1g piece add 300 uL buffer.
	#Transform 5-10 uL into 50 uL ~~DH5a~~ cells ~~(subcloning efficiency)~~:		:*Place at 55-65C until gel is dissolved (5-10 min)
			:*Add to pink purification column, wash and elute in 30 uL
			#Combine 6 uL insert with 1 uL vector. Place at 50-65C for 5-10 min to help sticky end binding.
			#Add 2 uL ligase buffer (single use aliquots).
			#Add 1 uL T4 DNA Ligase (Invitrogen).
			#Incubate 2h-O/N at 16C (water bath in cold room).
			#Transform 5-10 uL into 50 uL supercompetent cells:
	##Make 50 uL aliquots		##Make 50 uL aliquots
	##Add DNA and mix gently		##Add DNA and mix gently
	##Incubate on ice for 30min		##Incubate on ice for 30min
	##Heat shock for ~~20s~~ at ~~37C~~		##Heat shock for 45s at 42C
	##Place on ice for 2min		##Place on ice for 2min
	##Add 450 uL LB		##Add 450 uL LB
	##Incubate at 37C for 1h with shaking		##Incubate at 37C for 1h with shaking
	##Plate 500 uL and grow O/N at 37C on appropriate plates		##Plate 500 uL and grow O/N at 37C on appropriate antibiotic plates
	#Pick several colonies and digest to verify insert. Sequence if necessary.		#Pick several colonies and digest to verify insert. Sequence if necessary.

Davebrid: copied over protocol

2009-05-02T00:31:25Z

copied over protocol

New page

#If insert is PCR, run PCR then PCR purify (Qiagen kit). Elute in 30 uL EB
#Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C. Add 1 uL of CIAP to the vector for the last ~10min at 37C.
#Gel purify both vector and insert (Qiagen kit). Elute in 30 uL
#Combine 3-6 uL insert with 1-2 uL vector. You want about a 3x excess of insert to vector. Place at 50-65C for 5-10 min to help sticky end binding. Also do a no insert negative control.
#Add 2 uL ligase buffer (single use aliquots) and water/EB to 10 uL final volume. Add 1 uL T4 DNA Ligase (Invitrogen).
#Incubate 1h-O/N at 16C (water bath in cold room).
#Transform 5-10 uL into 50 uL DH5a cells (subcloning efficiency):
##Make 50 uL aliquots
##Add DNA and mix gently
##Incubate on ice for 30min
##Heat shock for 20s at 37C
##Place on ice for 2min
##Add 450 uL LB
##Incubate at 37C for 1h with shaking
##Plate 500 uL and grow O/N at 37C on appropriate plates
#Pick several colonies and digest to verify insert. Sequence if necessary.