Team:Warsaw/Calendar-Main/20 June 2008

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

Piotr

  1. Result of ligation of pET15b-OmpA-alpha with protein Z DNA: 16 colonies grown.
  2. Each colony cultured overnight in LB + ampicilin.

Preparation of constructs with OmpA protein fusions

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.

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

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.





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