Preparation of pSB2K3 + _alpha

Michał K.

1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using LinLSXNE and AlfaPSpe primers (annealing temperature 58 °C; elongation length 60s) to obtain link_alpha fragment.
2. Gel electrophoresis and gel-out of proper band 600 bp - link_alpha PCR product.
3. Digest of isolated PCR product link_alpha with EcoRI and BcuI (BamHI buffer).
4. Clean-up of digested PCR product.
5. Overnight ligation of DNA fragments: 3kb(?????)+ link_alpha.

Preparation of BioBricks

Michał K.

1. Digest of 3kb(????)+pLac_OmpA_omega with BcuI and SacI (SacI buffer), dephosphorylation with CIAP.
2. PCR on alpha PCR product from 10 September using AlphaL+SacI and LinP_BS2 primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha2 fragment with proper restriction sites.
3. Gel electrophoresis and gel-out of proper band 600 bp for alpha PCR products and 3000 bp for 3kb(????)+pLac_OmpA_.
4. Digest of isolated PCR products: alpha2 fragment with SacI and BcuI (SacI buffer) and link_alpha with EcoRI and BcuI (BamHI buffer).
5. Clean-up of digested PCR products.
6. Inoculation of colonies from plate with ligation of 5kb (??????) + pT7_omega_link to liquid LB + kanamycin and RFP(?????) + pT7_alpha fragment to liquid LB + ampicillin.
7. Inoculation of few more colonies from old plate with ligation of 3kb (??????) + OmpA_omega_link to liquid LB + kanamycin.
8. Overnight ligation of DNA fragments: 3kb(?????)+ link_alpha; 3kb(????)_pLac_OmpA_ + alpha2; RFP(?????) (from 6 October) + AraC+pBAD+AID.