MutD₅ testing

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Preparation of BioBricks

Michał K.

1. Digest of Z-GeneArt and RFP(????)+deltaA plasmids with NdeI and BcuI (BamHI buffer).
2. Digest of RFP(????)+deltaA plasmid with NdeI and SacI (BamHI buffer).
3. Dephosphorylation (CIAP) of plasmids.
4. Gel electrophoresis of digested plasmids and gel-out of proper bands (Z - 200 bp and both RFP (??????)'s - 2200 bp).
5. Gel electrophoresis to estimate concentration of purified DNA from previous day.
6. Overnight ligation of isolated DNA fragments: RFP(??????) + Z and RFP(????) + OmpA_link (from 18 September).
7. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using OmLNXNEB and LinPBSNP primers (annealing temperature 58 °C; elongation length 90s) to obtain OmpA_omega fragment.
8. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using PlacL_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain pLac_OmpA_omega fragment.
9. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using AL_BNXNE and APSacSpe primers (annealing temperature 58 °C; elongation length 45s) to obtain deltaA fragment.
10. PCR on pACYC177 + OmpA_omega plasmid using OmegLNde and LinP_BS primers (annealing temperature 55 °C; elongation length 60s) to obtain omega_link fragment.
11. Gel electrophoresis of PCR products and gel-out of proper bands (deltaA - 250 bp, pLac_Omp_omega_ - 1200 bp, linker_omega - 350 bp and Omp_omega_ - 900 bp).

Piotr

Inoculation of bacteria received from iGEM HQs ( ).