Team:Warsaw/Calendar-Main/31 August 2009

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Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil


Tasks:

  • mgtC promoter digest
  • Ligation

Methods:

  • The digest mix was prepared as follows:

    40μl purified PCR product
    5μl Buffer O (Fermentas)
    1μl EcoRI enzyme
    1μl PstI enzyme
    3μl H2O
  • The digest was carried out in 37°C for 3h and inactivated for 15 min. in 80°C.
  • The product was later purified using Montage PCR Centrifugal Filter Device (Milipore).
  • The ligation mix was prepared as follows: 

    40μl purified plasmid backbone
    20μl purified PCR product
    8μl Tango Buffer (Fermentas)
    5μl 5mM dNTPs (EURx)
    1μl T4 Ligase (Fermentas)
    6μl H2O
  • The ligation was carried out in 18°C overnight and then inactivated in 80°C for 15 min.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil


Tasks:

  • Ligation to the plasmid

Methods:

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