Knight:Site-directed mutagenesis/Multi site
Back to Site-directed mutagenesis
Work in progress!
This protocol permits mutation at multiple sites simultaneously with only a single oligo per site.
- Template plasmid purified from a dam+ E. coli strain (not JM110 or SCS110)
- Mutatagenesis primers (one per mutation, each on the same strand)
- PfuTurbo DNA polymerase (nonstrand-displacing) and associated reaction buffer
- Taq ligase
- 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.
- 1 μL of dNTP mix (100mM total dNTP mix with 25 mM each individual dNTP)
- ddH2O to a final volume of 25 μL
- 1 μL of PfuTurbo DNA polymerase (2.5 U/μL)
- 1 μL of Taq Ligase
- 1 μL of T4 PNK
This procedure is primarily derived from the Stratagene QuikChange Multi Site-Directed Mutagenesis manual with some modifications based on past experience.
- 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.
- Purify template plasmid from a dam+ E. coli strain via miniprep.
- Set up mutagenesis PCR mix as described above.
- Run Reaction
- 37°C for 30 min (T4 PNK step)
- 95°C for 3 min
- 95°C for 1 min
- 55°C for 1 min
- 65°C for 2 min/kb of plasmid length minimum
- Run reaction for 30 cycles.
- Stratagene recommends using a PCR machine with heated lid or overlaying the reaction with mineral oil.
- Cool the reaction to <=37°C
- Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage).
- Incubate 6 hours at 37°C (even though the Stratagene manual only recommends 1 hr).
- 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.
- Transform purified DNA into highly competent cells.
- Screen the transformants for the desired mutation using colony PCR, restriction digest or sequencing as appropriate.
- 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).