Team:Warsaw/Calendar-Main/27 June 2008

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Change of the reporter from pZC320 with B-galactosidase to GFP or RFP

Piotr, Weronika

  1. Isolation of pSB1A2 standard plasmids carrying parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator).
  2. Digest of pSB1A2 standard plasmids carrying parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator) with NotI.
  3. Digest of pZC320 with NotI and dephosphorylation with CIAP.
  4. Gel electrophoresis and gel-out of proper bands.
  5. Ligation of pZC320 with standard parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator).
  6. Chemotransformation of E.coli TOP10 with ligation products.
  7. Plating transformants on LB+Amp30+X-gal+IPTG.
Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

Clean-up of OmpA_alpha and OmpA_omega digest products.

Cloning alpha-A and omega-A fusions on pKS

Michał L., Ewa, Marcin

  1. Gel electophoresis of PCL products (Fig. 1).
  2. Gel-out of proper bands (alpha-A: 1100 bp and omega-A: 750 bp).
  3. Restriction digest of pKS vector and the PCL products with NotI and SacI (BamHI buffer) for 2 hours.
  4. Ligation 1 hour.
  5. Transformation of Top10 strain and screening on Amp100 plates.
Fig. 1.PCL product - Alpha-A fusion.