Team:Warsaw/Calendar-Main/14 May 2008

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Preparation of pMPMT5+AID construct

Michał K.

  1. Isolation of hypothetical pMPM-T5+AID plasmids from cultures inoculated on previous day.
  2. Restriction digest of plasmids with HindIII and NcoI (1xTango buffer) - construct control.
  3. Gel electrophoresis - choice of proper clones (all checked colonies). Fig. 1.
Fig. 1. 1-DNA ladder; 2 to 9-plasmids isolated from colonies of transformants and digested with HindIII and NcoI

Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7

Michał K.

  1. Optimization of conditions for PCR - T7 RNA polymerase for translation fusion; annealing temperature gradient (from 62°C to 82°C); Taq polymerase with supplemented buffer (Łukasz Banasiak helped us with that problematic PCR).
    Primers: T7lLinkB T7pXbSal
    Template DNA: E. coli Rosetta genomic DNA
    Elongation time: 4 minutes
    20 cycles
  2. Gel electrophoresis of PCR products.

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