Team:Warsaw/Calendar-Main/16 June 2008

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<h3>Preparation of constructs with OmpA protein fusions</h3>
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<h4>Michał K.</h4>
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<h4>Michał K.:</h4>
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Revision as of 10:41, 6 October 2008

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Michał L., Ewa, Marcin:

  1. Colony PCR
    Template: DNA isolated from white colonies
    Primers: pZCseqL and pZCseqR
    Annealing time:
    Number of cycles:
  2. DNA gel electrophoresis of PCR products.
  3. Gel-out of proper products (~1200 bp).
  4. Sequencing of proper fragments using primer pZCseqL.

Preparation of constructs with OmpA protein fusions

Michał K.:

  1. PCR on pB30D plasmid with OmpaL_N and OmpaP_link primers (15 cycles, elongation duration 45 s, annealing temperature 63°C).
  2. PCR on pUC19 plasmid with AlphaL_link and AlphaP_XB primers (20 cycles, elongation duration 45 s, annealing temperature 63°C).
  3. PCR on pUC19 plasmid with OmegaL_link and OmegaP_EPB primers (20 cycles, elongation duration 30 s, annealing temperature 58°C).
    As a result we got three PCR products: OmpA_linker and two fragments of TEM1 beta-lactamese: linker_alpha and linker_omega.
  4. Gel electrophoresis of PCR products and gel-out of proper bands (OmpA_linker - 500 bp, linker_alpha - 600 bp and linker_omega - 350 bp).