I. Primer Design.
          * Size: 25 ~ 45bp, with 10-15 bp of matching sequence on each mutation side. All oligos should anneal to the same strand of the template DNA
          * Melting temperature: >=75°C, they should have comparable melting temperatures.
          * >40% GC content and terminate in one or more C or G bases at the 3′ end.
   II. Make mini-prep plasmid from a dam+ E. coli strain.
   III. Set up mutagenesis PCR mix:

    25μL total reaction volume:

    * 2.5 μL of 10X Taq ligase buffer.
    * 0.5 μL 100mM ATP.
    * 1 μL 25mM each dNTP.
    * X μL (50-100 ng) of dsDNA template.
    * X μL of each oligonucleotide primer, e.g., 0.4 μL of 10μM each primer.
    * 1 μL of 10 mM dNTP mix.
    * Add H2O to a final volume of 22 μL.
    * 1 μL of PfuTurbo DNA polymerase (2.5 U/μL).
    * 1 μL of Taq Ligase.
    * 1 μL of T4 PNK.

   IV. PCR
         1, 37°C for 30 min (T4 PNK step)
         2, 95°C for 3 min
         25 cycles of step 3 to 5:
         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.  65°C for 5 min
   V. Cool the reaction to <=37°C
   VI. Add 1μL DpnI restriction enzyme.
   VII. Incubate 1 to 6 hours at 37°C.
   VIII. Purify PCR product using Qiagen PCR purification kit and transform Top10 competent cells.