Team:TU Darmstadt/safety/Labjournal

Assembly of the gBlocks

Based on the pDawn Plasmid we desinged the light induced kill switch and synthesized the construct as gBlock Fragments on IDT. We assembeled the 10 Fragments follow the Protocol:

• Reconstitute the gBlock Fragments in 10 µl TE Buffer
• Use 1 µl of each gBlock Fragment for a PCR with the Q5 Polymerase
• Perform the PCR Reaktion with the Primers Prefix and Suffix and an annealing temperature of 55 °C
  (30 cycles)
• Take 5 µl of the 50 µl reaction for DNA-gelelectrophoreses
• Restrict the other 45 µl with the restrictionenzymes EcoRI and PstI (10 U each) for 1 h at 37 °C
• Ligate 5 µl of the reaction mix with 50 ng EcoRI/PstI resticted and purified pSB1C3 over night at 16 °C
  For Ligation use the T4-Ligase and fresh T4-Ligase Buffer with ATP
• After heat-inactivation of the ligase by 80 °C for 15 min use 2 µl of the mix for heat-shock transformation
• Plate out the transformation in black petridishies