Preparation of T7 constructs
Piotr

1. DNA (PCR products from previous day) gel electrophoresis and isolation of DNA from proper bands.
2. Digest of DNA with HindIII and SalI (2x Tango buffer).
3. Digest of pMPM-T5 + AID with HindIII and SalI.
4. Clean-up of reaction products.
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. Plating transformants on LB+tetracycline.
8. Digest of pMPM-T5 + transcription fusion with EcoRHI and HindIII.
9. Use of T4 polymerase for blunting.
10. DNA gel electrophoresis.
11. Isolation of DNA from proper band.
12. Ligation of product p.11 (pMPM-T5 + T7 RNA-polymerase).
13. Transformation of E.coli TOP10 with ligation product.
14. Plating transformants on LB + tetracycline.