Open main menu

Changes

Mutagenesis

178 bytes added, 15:15, 13 January 2010
updated for new PCR program and to use Pfu Turbo as the polymerase
*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
*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
*DpnI
##5 uL of dNTP mix
##28 uL of water
#Add 1 uL of Pfu Ultra HF PolymeraseTurbo#Run PCR Program (DAVE-MUTMutagenesis 8/15/20 minute, depending on template)
##95C for 30s
##Repeat 18 cycles:
##95C for 30s
##55C for 1 min
##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
#Add 1 uL DpnI, mix and spin down. Digest 1h or overnight at 37C.