930
edits
Changes
copied over protocol
==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
*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
