Preparation of T7 constructs

Michał K.:

1. DNA (PCR products from previous day) gel electrophoresis and isolation of DNA from proper band.
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.