WangLab:Deletion or Insertion of Amino Acids w/o the Involvement of RE sites

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Deletion or Insertion of amino acids without the involvement of restriction enzyme sites

  • Design primers as indicated:

Image:Deletion.bmp Image:Insertion.bmp

  • Run PCR as following (strategene mutagenesis kit):
10x buffer: 2.5 ul
DNA: 1 ul (prediluted 1:5)
Primer1: 0.625 ul (stock solution 100ng/ul)
Primer2: 0.625 ul (stock solution 100ng/ul)
H2O: 19.25 ul
DNTP: 0.5 ul
Pfu Turbo: 0.5 ul
  • Digest parental DNA with 0.5 ul DPNi for >2hr 37oC.
  • Gel purify the PCR product, do blunt ligation:
5x buffer: 4 ul
DNA: 5 ul
Ligase: 1 ul
H2O: 10 ul
  • Do transformation
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