Mutagenesis: Difference between revisions
→Protocol: added link to checking clones |
updated for new PCR program and to use Pfu Turbo as the polymerase |
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*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 | ||
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##5 uL of dNTP mix | ##5 uL of dNTP mix | ||
##28 uL of water | ##28 uL of water | ||
#Add 1 uL of Pfu | #Add 1 uL of Pfu Turbo | ||
#Run PCR Program ( | #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. | ||