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
Mutagenesis PCR mix
- 5 μL of 10X reaction buffer (comes with PfuTurbo DNA polymerase)
- X μL (5–50 ng) of dsDNA template
- X μL (125 ng) of each oligonucleotide 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 50 μL
- 1 μL of PfuTurbo DNA polymerase (2.5 U/μL)
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 PCR
- 95°C for 2 mins
- 95°C for 30 secs
- 55°C for 1 min
- 68°C for 1 min/kb of plasmid length minimum
- Run PCR for 18 cycles for point mutations (even though the Stratagene manual recommends 12, I've found 18 to be preferable).
- 68°C for 20 mins
- Stratagene recommends using a PCR machine with heated lid or overlaying the reaction with mineral oil.
- Note that it is pretty common to not be able to visualize the PCR product on a gel but yet have the mutagenesis still work.
- Cool the reaction to <=37°C
- Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage).
- Incubate 2-3 hours at 37°C (even though the Stratagene manual only recommends 1 hr).
- Purify PCR product.
- 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. Typically 1/4 or 1/8 colonies are correct.
- 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).