Team:Warsaw/Calendar-Main/16 June 2008

From 2008.igem.org

Revision as of 15:27, 24 October 2008 by Smaegol (Talk | contribs)

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
next notebook entry

 


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.


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.