Mutagenesis - Revision history

Davebrid: updated for new PCR program and to use Pfu Turbo as the polymerase

2010-01-13T15:15:53Z

updated for new PCR program and to use Pfu Turbo as the polymerase

← Older revision		Revision as of 15:15, 13 January 2010
Line 2:		Line 2:
	*Template (dilute to ~ 0.1 mg/mL		*Template (dilute to ~ 0.1 mg/mL
	*Primers (prepare by adding 2 uL of each sense and antisense primer + 196 uL water and label as 1 uM primer mix). Design using Stratagene Primer Design Program		*Primers (prepare by adding 2 uL of each sense and antisense primer + 196 uL water and label as 1 uM primer mix). Design using Stratagene Primer Design Program
			*dNTP's (2 mM; add 10 uL of each dNTP (100 mM from invitrogen) into 460 uL water, and make 50 uL aliquots)
	*PFU Turbo		*PFU Turbo
	*DpnI		*DpnI
Line 13:		Line 14:
	##5 uL of dNTP mix		##5 uL of dNTP mix
	##28 uL of water		##28 uL of water
	#Add 1 uL of Pfu ~~Ultra HF Polymerase~~		#Add 1 uL of Pfu Turbo
	#Run PCR Program (~~DAVE-MUT~~)		#Run PCR Program (Mutagenesis 8/15/20 minute, depending on template)
	##95C for 30s		##95C for 30s
	##Repeat 18 cycles:		##Repeat 18 cycles:
	##95C for 30s		##95C for 30s
	##55C for 1 min		##55C for 1 min
	##68C for 1 min/kb plasmid length (8 min default)		##68C for 1 min/kb plasmid length (8 min default, but use 15 or 20 min for larger templates.)
	#Place on ice for 2 min		#Place on ice for 2 min
	#Add 1 uL DpnI, mix and spin down. Digest 1h or overnight at 37C.		#Add 1 uL DpnI, mix and spin down. Digest 1h or overnight at 37C.

Davebrid: /* Protocol */ added link to checking clones

2009-05-27T13:06:24Z

Protocol: added link to checking clones

← Older revision		Revision as of 13:06, 27 May 2009
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	#Spread out entire transformation on appropriate antibiotic		#Spread out entire transformation on appropriate antibiotic
	#Grow O/N at 37C		#Grow O/N at 37C
	#Pick a colony, miniprep and sequence to verify mutation		#Pick a colony, miniprep and sequence to verify mutation
			#[[Check Clones for Correct Mutation]]

Davebrid: /* Protocol */

2009-05-02T00:29:53Z

Protocol

← Older revision		Revision as of 00:29, 2 May 2009
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	##68C for 1 min/kb plasmid length (8 min default)		##68C for 1 min/kb plasmid length (8 min default)
	#Place on ice for 2 min		#Place on ice for 2 min
	#Add 1 uL DpnI, mix and spin down. Digest 1h at 37C.		#Add 1 uL DpnI, mix and spin down. Digest 1h or overnight at 37C.
	#Thaw XL1-Blue cells on ice and make 50 uL aliquots (round tube)		#Thaw XL1-Blue cells on ice and make 50 uL aliquots (round tube)
	##Add 1 uL of digest to cells and mix.		##Add 1 uL of digest to cells and mix.

Davebrid: /* Protocol */

2009-05-02T00:29:18Z

Protocol

← Older revision		Revision as of 00:29, 2 May 2009
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	==Protocol==		==Protocol==
	#Mix:		#Mix:
	**5 uL 10X Reaction Buffer		##5 uL 10X Reaction Buffer
	**~20 ng Template (1 uL of minprep)		##~20 ng Template (1 uL of minprep)
	**10 uL of Primer Mix		##10 uL of Primer Mix
	**5 uL of dNTP mix		##5 uL of dNTP mix
	**28 uL of water		##28 uL of water
	#Add 1 uL of Pfu Ultra HF Polymerase		#Add 1 uL of Pfu Ultra HF Polymerase
	#Run PCR Program (DAVE-MUT)		#Run PCR Program (DAVE-MUT)

Davebrid: copied over protocol

2009-05-02T00:28:40Z

copied over protocol

New page

==Materials==
*Template (dilute to ~ 0.1 mg/mL
*Primers (prepare by adding 2 uL of each sense and antisense primer + 196 uL water and label as 1 uM primer mix). Design using Stratagene Primer Design Program
*PFU Turbo
*DpnI
*Supercompetent XL1-Blue Cells (Stratagene)

==Protocol==
#Mix:
**5 uL 10X Reaction Buffer
**~20 ng Template (1 uL of minprep)
**10 uL of Primer Mix
**5 uL of dNTP mix
**28 uL of water
#Add 1 uL of Pfu Ultra HF Polymerase
#Run PCR Program (DAVE-MUT)
##95C for 30s
##Repeat 18 cycles:
##95C for 30s
##55C for 1 min
##68C for 1 min/kb plasmid length (8 min default)
#Place on ice for 2 min
#Add 1 uL DpnI, mix and spin down. Digest 1h at 37C.
#Thaw XL1-Blue cells on ice and make 50 uL aliquots (round tube)
##Add 1 uL of digest to cells and mix.
##Incubate 30 min on ice.
##Heat at 42 C for 45 s, then place on ice for 2 min.
##Add 450 uL SOC preheated at 42C and grow 1h at 37C shaking
#Spread out entire transformation on appropriate antibiotic
#Grow O/N at 37C
#Pick a colony, miniprep and sequence to verify mutation