Team:Warsaw/Calendar-Main/16 May 2008

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PCRs for fusions

Michał K.:

  1. Gel electrophoresis (15 May PCR's) and DNA isolation from proper bands (600 bp for AID lane and 2700 bp for T7 polymerase lanes).

  2. PCR - translation fusion: AID + T7 RNA-polymerase - optimization (annealing temperature gradient 60°C - 80°C).

    Primers:

    AIDlNrH and T7pXbSal
    Template DNA: purified PCR products from 16 May - AID and T7 RNA-polymerase for translation fusion
    Elongation time: 4 minutes
    35 cycles

  3. Optimization of PCR - translation fusion: AID + T7 RNA-polymerase - MgCl2 cocentration and number of cycles.

    Primers:

    AIDlNrH and T7pXbSal

    Template DNA: purified PCR products from 16 May - AID and T7 RNA-polymerase for translation fusion
    Annealing temperature: 73°C
    Elongation time: 4 minutes
  4. Gel electrophoresis of PCR products.