Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7

Michał K.:

1. Digest of PCR product for transcription fusion (T7 RNA-polymerase) with HindIII and SalI (2x Tango buffer).
2. Digest of PCR product for translation fusion (AID+T7 RNA-polymerase) with NcoI and XbaI (1x Tango buffer).
3. Clean-up of digested products from p.1 and p.2.
4. Digest of pMPM-T5+AID with HindIII and SalI (2x Tango buffer), dephosphorylation (CIAP).
5. Digest of pMPM-T5 with NcoI and XbaI (1x Tango buffer), dephosphorylation (CIAP).
6. Gel-electrophresis and gel-out of proper bands (p.4 - 4850 bp, p.5 - 4250 bp).
7. Electrophoresis to estimate the concentration of purified DNA.
8. Ligation of purified products from p.1 and p.4.
9. Electroporation of E. coli TOP10 with ligation product.
10. Ligation of digested products from p.4 and p.5.
11. Electroporation of E. coli TOP10 with ligation product.
12. Transformants plating on LB + tetracycline.

Rifampicin test

Michał L., Ewa, Marcin:

1. Plating colonies from the previous day on rifampicin.