Team:Warsaw/Calendar-Main/10 October 2008

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Visit in US Embassy

Visas have been accorded to the whole team.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K., Piotr

  1. Clean-up of overnight digest reaction.
  2. Digest of pSB1A3 carrying ΔA (BBa_K103003) with EcoRI and SacI (BamHI buffer), dephosphorylation with CIAP
  3. Gel electrophoresis and gel-out of proper band - 2200 bp.
  4. Ligation of digested pSB1A3 with alpha_linker under PT7 (BBa_K103019) fragment (1 hr).
  5. Transformation of TOP10 with above ligation.
  6. Tranformants plating on LB with ampicillin.

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K., Piotr

  1. Ligation of digested pSB2K3 vector from (30 September) with omega_linker under PT7 (BBa_K103020) fragment (1 hr).
  2. Transformation of TOP10 with above ligation.
  3. Tranformants plating on LB with kanamycin.

Preparation of AID under pBAD/araC (BBa_K103002)

Michał K., Piotr

  1. Isolation of plasmid from culture inoculated on previous day - pMPMT5+AID (with removed EcoRI site).
  2. Control digest of isolated plasmids with EcoRI and BamHI (BamHI buffer) - proper clones found.
  3. Temperature gradient PCR on pMPMT5+AID (with removed EcoRI site) plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 40 - 60 °C; elongation length 2.30 min) to optimize conditions for obtaining AID under pBAD/araC (BBa_K103002).
  4. Gel electrophoresis. Best annealing temperature (45 °C) chosen
  5. PCR on pMPMT5+AID (with removed EcoRI site) plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 45 °C; elongation length 2.30 min) to obtain AID under pBAD/araC (BBa_K103002)fragment.
  6. Gel electrophoresis and gel-out of proper band 1800 bp.
  7. Digest of isolated PCR product with XbaI and PstI (Tango buffer).
  8. Clean-up of digested PCR product.

Preparation of OmpA_linker_alpha_linker under Plac (BBa_K103017)

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using AlphaL+Nde and AlphaP+link10+homo2 primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha fragment.
  2. Gel electrophoresis and gel-out of proper band 600 bp.
Fig. 9.Go_10_10_2008.jpg