Team:Warsaw/Calendar-Main/20 June 2008

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Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

  1. Isolation of plasmids from cultures inoculated on previous day.
  2. Control digest of isolated plasmids with PstI (Orange buffer).
  3. Gel electrophoresis (Fig. 1 and 2), no proper colonies found.
Fig. 1. Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_alpha fusion with PstI. Fig. 2. Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_omega fusion with PstI.

Preparation of alpha-A and omega-A fusions

Michał L., Ewa, Marcin

PCR
ProductTemplatePrimersProduct length
linker-ApDRIVE-TapTagAL+link10+homo2 and AP+NotI 470 bp
alpha-linkerpUC19 AlphaL+SacI and AlphaP+link10+homo2600 bp
omega-linkerpUC19OmegaL+SacI and OmegaP+link10+homo2400 bp



Universal PCR program for protein A, alpha and omega
TemperatureTime
94°C4:00
94°C0:3028 cycles
50°C0:45
72°C2:00
72°C10:00
4°Cinfinite

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