Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/07/15

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Miniprep liquid cultures of pLux1, 2, 3 and LasR 1. Follow up with a XbaI + KpnI digestion; if there was an insertion, the KpnI site would be gone, resulting in one band. If there was no insertion, and the KpnI site was still there, there would be two bands.

File:15.07.15 yaaaaay luxR lasR inv rep insertions digest ver.tiff

Unsure why there are three bands for LasR1 and pLux1. pLux 2 and 3, however, look promising, so they will be sent in for sequencing.

Troubleshooting possibilities:

  • MRV was not treated with SAP (Shrimp Alkaline Phosphatase), so it may have re-ligated to itself, resulting in the copious amount of colonies on the backbone control plate. Solution: treat MRV with SAP
  • LasR backbone showed two bands...there may have been contamination during the previous gel extraction. Solution: Re-try the gel extraction