Site-directed mutagenesis/Multi site-PDF

Materials

• Template plasmid purified from a dam⁺ E. coli strain (not JM110 or SCS110)
• Mutagenesis primers (one permutation, each on the same strand)
• PfuTurbo DNA polymerase (non strand-displacing) and associated reaction buffer
• Taq ligase
• dNTPs
• ATP
• PCR Thermocycler
• Dpn I
• Competent cells
• unphosphorylated primers (1 for each mutation)

Mutagenesis PCR mix

25μL total reaction volume:

• 2.5 μL of 10X Taq ligase buffer (need the NAD for Taq ligase)
• 0.5 μL 100mM ATP
• 1 μL 25mM each dNTP
• X μL (50-100 ng) of dsDNA template
• X μL of each oligonucleotide primer
  • For 1-3 primers, add 100 ng each primer. For 4-5 primers, add 50 ng each primer.
  • If primers are greater than 20% different in length, scale the amount of primer added so that primer is added in approximately equimolar amounts. See Stratagene QuikChange Multi Site-Directed Mutagenesis manual for details.
  • Austin uses 0.1μL of 40μM primer (each one).
• 1 μL of dNTP mix (100mM total dNTP mix with 25 mM each individual dNTP)
• ddH₂O to a final volume of 22 μL

Then add

• 1 μL of PfuTurbo DNA polymerase (2.5 U/μL)
• 1 μL of Taq Ligase
• 1 μL of T4 PNK

Procedure

This procedure is primarily derived from the Stratagene QuikChange Multi Site-Directed Mutagenesis manual with some modifications based on past experience.

1. Design mutagenesis primers.
   • The primer should be designed so that the desired mutation occurs at the exact center of the primer with 10-15 bp of matching sequence on each side.
   • Primers should be 25-45bp in length with a melting temp of >=75°C. Stratagene recommends not using primers greater than 45 bp in order to avoid formation of secondary structure. Primers should have comparable melting temperatures.
   • See the Stratagene manual for more detailed information. In particular, adhere to their formula for calculating the melting temperature of your primers and design your primers to have a melting temperature >=75°C.
   • Primers should have at least 40% GC content and terminate in one or more C or G bases at the 3′ end.
   • PAGE purification of primers may improve mutagenesis efficiency. See here for links to information on oligo purification.
   • See designing primers for general advice on primer design.
2. Purify template plasmid from a dam⁺ E. coli strain via miniprep.
3. Set up mutagenesis PCR mix as described above.
4. Run Reaction
   1. 37°C for 30 min (T4 PNK step)
   2. 95°C for 3 min
   3. 95°C for 1 min
   4. 55°C for 1 min
   5. 65°C for 2 min/kb of plasmid length minimum (is optimal temperature for Taq ligase)
   6. Run reaction for 30 cycles.
   • Stratagene recommends using a PCR machine with heated lid or overlaying the reaction with mineral oil.
5. Cool the reaction to <=37°C
6. Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage).
7. Incubate 6 hours at 37°C (even though the Stratagene manual only recommends 1 hr).
8. Purify PCR product (not necessary, Austin transforms 3 μl directly).
   • I typically do this step using a QIAgen PCR Purification kit but any purification which removes the salts, dNTPs, oligos and proteins from the PCR should be fine.
9. Transform purified DNA into highly competent cells.
10. Screen the transformants for the desired mutation using colony PCR, restriction digest or sequencing as appropriate.

Notes

• Stratagene does not recommend this protocol for insertions or deletions.
• Apparently there are no primer spacing-dependent effects on mutagenesis efficiency (primers can be adjacent or far apart).
• You can also phosphorylate the primers separately from the rest of the mutagenesis reaction