Insertion of the pho gene into the pKSII+ plasmid

Kama

• Plasmid digest mix was prepared as follows:

  2μl Tango buffer (Fermentas)
  1μl pKSII plasmid
  1μl XbaI enzyme
  1μl SmaI enzyme

  the solution was topped up with H2O to the final volume of 20 μl.
• Pho gene digest mix was prepared as follows:

  2μl Tango buffer (Fermentas)
  3μl purified gene
  1μl XbaI enzyme

  the solution was topped up with H2O to the final volume of 20 μl.
• The digest was kept for 3h at 37°C, and then the enzymes were inactivated for 20min. at 80°C.

• The ligation mix was prepared as follows:

  20μl plasmid
  20μl gene
  5μl ligation buffer G (Fermentas)
  2μl T4 DNA ligase (Fermentas)

  the solution was topped up with H2O to the final volume of 50 μl. The ligation was carried out in 18°C overnight (~15h)