Creation of the cro-box testing devices

Kamil

Tasks:

• Reporter device carrying plasmid digestion
• Cro-box carrying plasmids digestion

Methods:

• For the reporter device the digest mix was prepared as follows:

  10μl purified plasmid 
  2μl Tango Buffer (Fermentas)
  1μl XbaI Enzyme (Fermentas)
  1μl PstI Enzyme (Fermentas)
  5μl H2O

• For the cro-box the digest mix was prepared as follows (same for both plasmids):

  10μl purified plasmid 
  2μl Tango Buffer (Fermentas)
  1μl BcuI Enzyme (Fermentas)
  1μl PstI Enzyme (Fermentas)
  5μl H2O

• The digest was carried out in 37°C for 4h.
• The results were visualized on 1% agarose gel.
• Gel extraction and purification of the appropriate bands was carried out using the Gel-Out kit (A&A Biotechnology)

Mutagenesis of cI

Kuba

Task: Obtain a functional, termosensitive version of cI

• inactivation ligation (15', 65^oC)
• Dpn1 digest (5 h)
• transformation into E. coli (Top10)