Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2016/02/02

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02/02/2016

Ligating LasR_MRV Backbone with gB015 Insert

Jiaqi and I performed ligations with a LasR_MRV backbone and a gB015 insert. The resulting plasmid from the ligation was a positive control. This means the plasmid has a constitutive promoter, which causes GFP to be produced continuously without needing to be triggered by a signaling molecule. Four different samples were ligated under the presence of the enzyme ligase:

  1. Backbone from PCR purification + insert
  2. Backbone from digestion + insert
  3. Backbone only control
  4. Negative control

The backbone control was performed to make sure the vector did not re-anneal to itself and leave out the insert, and the negative control was used to check for contamination.

To allow transformation to occur, 45 μL of cells were added to 5 μL of plasmid for samples 1 through 3, with the plasmid replaced with an equivalent amount of water for the negative control. The transformed samples were then incubated in agar plates for about 18 hours at 37°C. Plates 1 and 2 exhibited an abundance of colonies the next day, with only 9 on the backbone control. The negative control showed that the samples were free of contamination, as it was completely clean.