WangLab:Deletion or Insertion of Amino Acids w/o the Involvement of RE sites
From OpenWetWare
Deletion or Insertion of amino acids without the involvement of restriction enzyme sites
- Design primers as indicated:
- 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
