Team:Warsaw/Calendar-Main/3 June 2008

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Preparation of pMPMT5-AID+AIDT7 construct

Michał K.

DNA (PCR products from previous day) gel electrophoresis and isolation of DNA from proper band (20 cycles - 3300 bp). Fig. 1.

Fig. 1. PCR results of translational fusion for the pMPMT5-AID+AIDT7 construct.
Lane 1 - 25 cycles, lane 2 - 20 cycles.

Preparation of pMPMT5+T7 construct

Piotr, Weronika

  1. Digest of pMPMT5-AID+T7 (transcriptional fusion) with EcoRI and HindIII (2x Tango buffer).
  2. Use of T4 polymerase for blunting (3 hr of incubation).
  3. DNA gel electrophoresis.
  4. Isolation of DNA from proper band (7000 bp).
  5. Ligation of isolated DNA (pMPM-T5 + T7 RNA-polymerase).
  6. Transformation of E.coli TOP10 with ligation product.
  7. Plating transformants on LB + tetracycline.

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