Team:Warsaw/Calendar-Main/14 August 2009

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Cloning Bax into pBS plasmid

Justyna

Task 1:

  • Gel-out Bax PCR product

Methods:

  • Bax PCR products were isolated from agarose gel using A&A Gel-Out kit.


Task 2:

  • Digestion and gel-out of pBS plasmid

Methods:

  • Digestion of isolated plasmids by XbaI and SpeI
  • Reaction mixture composition: 10.0 μl purified plasmid DNA product, 0.5 μl XbaI (Fermentas),0.5 μl SpeI (Fermentas), 2 μl Buffer Orange (Fermentas), 7.0 μl MQ water
  • The digestion was performed overnight and it was subsequently inactivated via heating in 80°C for 20 minutes.
  • In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids and isolated using A&A Gel-Out kit

Task 3:

  • Ligation of Bax and pBS

Methods:

  • The full description of ligation is here


Task 4:

  • Insertion of Bax-pBS plasmid into bacteria

Methods:

  • Previously prepared chemicompetent bacteria were used.
  • The full description of the procedure is here








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