Team:Cambridge/Lab book/Week 8


Revision as of 13:30, 30 August 2012 by Olijme (Talk | contribs)

Week: 3 4 5 6 7 8 9 10 11 12


Monday (13/08/12)

Tuesday (14/08/12)

Gibson assembly of positive control

  • Fragments used:
  • Protocol changed slightly: 0.5 μl of each DNA fragment solution mixed in with master mix to make up 4 μl total volume (1μl DNA and 3μl master mix).

Transformation of e.coli with positive control DNA

  • Chemically competent e.coli cells transformed with plasmid DNA produced by Gibson assembly step.
  • Transformants plated out on 50 μg/ml kanomycin plates. Incubated overnight at 37 °C.

Wednesday (15/08/12)

PCR of positive control fragments

  • Standard PCR settings used

Extraction of positive control DNA

  • Positive control DNA from gel excised and purified.
  • Additional elution steps used to concentrate resultant solution.
  • Verified with nanodropper - final DNA concentrations:

Gibson assembly of positive control DNA

Transformation of positive control DNA into e.coli

  • Chemically competent e.coli transformed with positive control Gibson products made previously.
  • Transformants plated out onto 50 μg/ml kanomycin plates and incubated at 37 °C overnight.

Characterization of fluoride riboswitch construct

  • Strain of bacillus lacking fluoride transporter system tested. Concentrations used:
  • 0mM - 0.5mM - 1mM - 2.5mM - 5mM - 10mM - 20mM - 30mM

Thursday (16/08/12)

Characterization of fluoride riboswitch construct

The results of our fluoride assay.
  • Eppindorfs containing the bacillus with the fluoride riboswitch removed from incubator and imaged.
  • Now this definitely works, we will try quantifying this riboswitch with an ONPG assay and the plate reader.

PCR ran by paul - insert photos/discuss

  • Only positive control and two sets of triplicates worked
  • YFP and CFP extracted successfully

Friday (17/08/12)

  • Team meet up today! No lab work was done.

Saturday (18/08/12)