Mutagenesis

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
• 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
• Supercompetent XL1-Blue Cells (Stratagene)

Protocol

1. Mix:
   1. 5 uL 10X Reaction Buffer
   2. ~20 ng Template (1 uL of minprep)
   3. 10 uL of Primer Mix
   4. 5 uL of dNTP mix
   5. 28 uL of water
2. Add 1 uL of Pfu Turbo
3. Run PCR Program (Mutagenesis 8/15/20 minute, depending on template)
   1. 95C for 30s
   2. Repeat 18 cycles:
   3. 95C for 30s
   4. 55C for 1 min
   5. 68C for 1 min/kb plasmid length (8 min default, but use 15 or 20 min for larger templates.)
4. Place on ice for 2 min
5. Add 1 uL DpnI, mix and spin down. Digest 1h or overnight at 37C.
6. Thaw XL1-Blue cells on ice and make 50 uL aliquots (round tube)
   1. Add 1 uL of digest to cells and mix.
   2. Incubate 30 min on ice.
   3. Heat at 42 C for 45 s, then place on ice for 2 min.
   4. Add 450 uL SOC preheated at 42C and grow 1h at 37C shaking
7. Spread out entire transformation on appropriate antibiotic
8. Grow O/N at 37C
9. Pick a colony, miniprep and sequence to verify mutation
10. Check Clones for Correct Mutation