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Colony PCR

This step takes place after having transformed cells with Gibson-assembled plasmids and having grown them overnight on a plate. It is quicker to check the plasmids at this stage rather than waiting for a miniprep.

• Recover the colonies:
  • Choose a few colonies that you want to analyze
  • Prepare PCR tubes with 50 ul water
  • For each colony, take it with a tip, then plate it on a new agar plate (just make a dot). You can use one plate for all the colonies tested.
  • Put the tip in a PCR tube and mix.
  • Boil the tubes at 95°C for 10min (use the "BOIL" file)

It is important to place the colonies on a new plate, because if the PCR gives good results we need the corresponding cells!

• Prepare the PCR: protocol adapted for HiFi PLUS enzyme, for 50 ul final volume
  • reaction buffer 5x (with MgCl2): 10 ul
  • dNTPs 10mM: 1 ul
  • each primer (20 uM): 1 ul (nneds to be 0.4 uM in final volume)
  • template DNA (boiled samples): 1ul
  • HiFi PLUS (5 U/ul): 0.5 ul
  • ddH20: 35.5 ul (check to have 50ul total)

• For J61002 Ptet-RFP plasmid (control to see if colony PCR is working):
  • use J6-Ptet_RFP-f and J6-Ptet_RFP-r, use the "VINCECOL" file
• For reporter plasmid:
  • use J6Ptet-LacI-RFPf and J6Ptet-LacI-RFPr
• For lysis plasmid:
  • use J6Ptet-LacI-RFPf and J6Ptet-LacI-Lysr