Michał K.:

1. Digestation of PCR product for transcription fusion (T7 RNA-polimerase) with HindIII and SalI (2x Tango buffer).

2. Digestation of PCR product for translation fusion (AID+T7 RNA-polimerase) with NcoI and XbaI (1x Tango buffer).

3. Clean-up of digested products from p.1 and p.2.

4. Digestation of pMPM-T5+AID with HindIII and SalI (2x Tango buffer).

5. Digestation of pMPM-T5 with NcoI and XbaI (1x Tango buffer).

6. Gel-electrophresis and gel-out of proper bands (p.4 - 4850 bp, p.5 - 4250 bp).

7. Ligation of purified products from p.1 and p.4

8. Electroporation of E.coli TOP10 with ligation product.

9. Ligation of digested products from p.4 and p.5.

10. Electroporation of E.coli TOP10 with ligation product.

11. Transformants plating on LB + tetracycline<./p>

http://openwetware.org/images/thumb/6/61/UW_A%2BT.jpg/350px-UW_A%2BT.jpg http://openwetware.org/images/thumb/c/c7/UW_AT.jpg/350px-UW_AT.jpg