IGEM:UW/2008/Notebook/iGEM UW Team/2008/06/03

1. Gel electrophoresis (PCR products) and isolation from proper bands

2. Digest with HindIII and SalI

3. Digest of pMPM-T5 + AID with HindIII and SalI

4. Clean-up products of p.2 and p.3 reactions

5. Ligation of p.4 products (pMPM-T5 + AID transcription fusion with AID-T7 RNA-polymerase translation fusion)

6. Transformation of E.coli TOP10 with ligation product

7. Transformants onto LB+tetracycline

8. Digest of pMPM-T5 + transcription fusion with EcoRHI and HindIII

9. Use of T4 polymerase for blunting

10. Gel electrophoresis

11. Isolation from proper band

12. Ligation of product p.11 (pMPM-T5 + T7 RNA-polimerase)

13. Transformation of E.coli TOP10 with ligation product

14. Transformants onto LB + tetracycline