Team:Warsaw/Calendar-Main/16 June 2008

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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 (Fig. 1) and gel-out of proper bands (OmpA_linker - 500 bp, linker_alpha - 600 bp and linker_omega - 350 bp).
  5. Electrophoresis to estimate the concentration of isolated DNA.

Blue/white test

Michał L., Ewa

  1. Colony PCR.
    Template: DNA isolated from white colonies
    Primers: pZCseqL and pZCseqR

    Colony PCR program
    TemperatureTimeNo. of cycles
    94°C4:00
    94°C0:3028 cycles
    48°C0:45
    72°C1:30
    72°C10:00
    4°Cinfinite

  2. DNA gel electrophoresis of PCR products. Fig. 2.
  3. Gel-out of proper products (~1200 bp).
  4. Sequencing of proper fragments using primer pZCseqL.

Cloning of protein Z DNA to pET15b-OmpA-alpha in place of OmpA

Piotr, Weronika

  1. Digest of pET15b-OmpA-alpha and Z (in Geneart vector) with NdeI and NotI (Orange buffer).
  2. Gel-out of Z (~200 bp band).
  3. Overnight ligation of Z into digested pET15b-OmpA-alpha.

Fig. 1. PCR products: linker_alpha (lane 2), linker_omega (lane 3), OmpA_linker (lane 4) Fig. 2. 1 - DNA ladder; 2 to 11 - colony PCR products (lacZ' gene) from blue and white colonies.