Team:Cambridge/Lab book/Week 8

From 2012.igem.org

(Difference between revisions)
(Wednesday (15/08/12))
(Thursday (16/08/12))
Line 63: Line 63:
===Thursday (16/08/12)===
===Thursday (16/08/12)===
 +
 +
'''[[Team:Cambridge/Protocols/beta-galactosidaseassay| Characterization of fluoride riboswitch construct]]'''
 +
 +
----
 +
 +
[[File:fluoridegradient.jpg|250px|right|thumb|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.
===Friday (17/08/12)===
===Friday (17/08/12)===
{{Template:Team:Cambridge/CAM_2012_TEMPLATE_FOOT}}
{{Template:Team:Cambridge/CAM_2012_TEMPLATE_FOOT}}

Revision as of 11:25, 16 August 2012

Week: 3 4 5 6 7 8 9

Contents

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:

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.

Friday (17/08/12)