Knight:Site-directed mutagenesis/Multi site

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This protocol permits mutation at multiple sites simultaneously with only a single oligo per site.

Materials

 * 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
 * dNTPs
 * ATP
 * PCR Thermocycler
 * Dpn I
 * Competent cells
 * unphosphorylated primers (1 for each mutation)

Mutagenesis PCR mix
25&mu;L total reaction volume: Then add
 * 2.5 &mu;L of 10X Taq ligase buffer (need the NAD for Taq ligase)
 * 0.5 &mu;L 100mM ATP
 * 1 &mu;L 25mM each dNTP
 * X &mu;L (50-100 ng) of dsDNA template
 * X &mu;L 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 0.1&mu;L of 40&mu;M primer (each one).
 * 1 &mu;L of dNTP mix (100mM total dNTP mix with 25 mM each individual dNTP)
 * ddH2O to a final volume of 22 &mu;L
 * 1 &mu;L of PfuTurbo DNA polymerase (2.5 U/&mu;L)
 * 1 &mu;L of Taq Ligase
 * 1 &mu;L 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.
 * 2) *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.
 * 3) *Primers should be 25-45bp in length with a melting temp of >=75&deg;C. Stratagene recommends not using primers greater than 45 bp in order to avoid formation of secondary structure.  Primers should have comparable melting temperatures.
 * 4) *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&deg;C.
 * 5) *Primers should have at least 40% GC content and terminate in one or more C or G bases at the 3' end.
 * 6) *PAGE purification of primers may improve mutagenesis efficiency. See  here for links to information on oligo purification.
 * 7) *See designing primers for general advice on primer design.
 * 8) Purify template plasmid from a dam+ E. coli strain via miniprep.
 * 9) Set up mutagenesis PCR mix as described above.
 * 10) Run Reaction
 * 11) 37&deg;C for 30 min (T4 PNK step)
 * 12) 95&deg;C for 3 min
 * 13) 95&deg;C for 1 min
 * 14) 55&deg;C for 1 min
 * 15) 65&deg;C for 2 min/kb of plasmid length minimum (is optimal temperature for Taq ligase)
 * 16) Run reaction for 30 cycles.
 * 17) *Stratagene recommends using a PCR machine with heated lid or overlaying the reaction with mineral oil.
 * 18) Cool the reaction to <=37&deg;C
 * 19) Add 1&mu;L DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage).
 * 20) Incubate 6 hours at 37&deg;C (even though the Stratagene manual only recommends 1 hr).
 * 21) Purify PCR product (not necessary, Austin transforms 3 &mu;l directly).
 * 22) *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.
 * 23) Transform purified DNA into highly competent cells.
 * 24) Screen the transformants for the desired mutation using colony PCR,  restriction digest or  sequencing as appropriate.

Reference
QuikChange® Multi Site Directed Mutagenesis Kit