Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/07/13

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07/13/2015

LasR

pLas has been double digested with EcoRI and SpeI, then heat inactivated using an annealing oligos protocol.
LasR + MRV has been digested with BbsI and gel-extracted for a gel purification.
Gel-purify LasR + MRV using Sigma Aldrich Gel Extraction Kit
Ligate pLas and LasR + MRV
Reagent Vol (uL)
Backbone 2
Insert 1
2x Lig Buffer 5
T4 Ligase 1
Water 1
Total 10
Transform with 50μL of DH5αT
  • In the backbone-only control, no insert was added; volume of water increased to make up for insert volume.

LuxR

pLux oligos have been annealed
  • 1uL each (top and bottom) at 100μM concentrations
Modular receiver vector was digested with BbsI, then PCR purified
Ligate pLux and MRV:
Reagent Vol (uL)
Backbone 2.7
Insert 1
2x Lig Buffer 5
T4 Ligase 1
Water 1.3
Total 11
Transform with 50μL of DH5αT
  • In the backbone-only control, no insert was added; volume of water increased to make up for insert volume.