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
• PFU Turbo
• DpnI
• Supercompetent XL1-Blue Cells (Stratagene)

Protocol

1. 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

1. Add 1 uL of Pfu Ultra HF Polymerase
2. Run PCR Program (DAVE-MUT)
   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)
3. Place on ice for 2 min
4. Add 1 uL DpnI, mix and spin down. Digest 1h at 37C.
5. 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
6. Spread out entire transformation on appropriate antibiotic
7. Grow O/N at 37C
8. Pick a colony, miniprep and sequence to verify mutation