Preparation of T7 constructs

By Michał K.

• DNA (PCR products from previous day) gel electrophoresis and isolation of DNA from proper bands.
• Digestion of DNA with HindIII and SalI.
• Digestion of pMPM-T5 + AID with HindIII and SalI.
• Clean-up of reaction products.
• Ligation of p.4 products (pMPM-T5 + AID transcription fusion with AID-T7 RNA-polymerase translation fusion).
• Transformation of E.coli TOP10 with ligation product.
• Plating transformants on LB+tetracycline.
• Digestion of pMPM-T5 + transcription fusion with EcoRHI and HindIII.
• Use of T4 polymerase for blunting.
• DNA gel electrophoresis.
• Isolation of DNA from proper band.
• Ligation of product p.11 (pMPM-T5 + T7 RNA-polymerase).
• Transformation of E.coli TOP10 with ligation product.
• Plating transformants on LB + tetracycline.