Team:Cambridge/Lab book/Week 8

From 2012.igem.org

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*DNA extracted and purified.
*DNA extracted and purified.
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'''[[Team:Cambridge/Protocols/PCRcolonyl|PCR of Mg2+ riboswitch construct vector]]'''
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'''[[Team:Cambridge/Protocols/PCRcolony|PCR of Mg2+ riboswitch construct vector]]'''
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:*Elongation - 72 &degC - 100 seconds  
:*Elongation - 72 &degC - 100 seconds  
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*Productos
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*Products run on gel. No fragments produced. Positive control worked however, so master mix clearly works.
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*Given this PCR has worked in the past, will try to re-run with the same settings and hope for success in the future.
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Revision as of 16:41, 31 August 2012

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

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:

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)

PCR of Fluoride biobrick format


Gels from PCR of Fluoride BB and PJS130 fragments. Top: Lanes 2-4: Gibson compatable Fluoride biobrick fragment. Lanes 5-7: Ligation compatable Fluoride biobrick fragment. Lane 8: PJS130 fragment A. Bottom: Lanes 1-2: PJS130 fragment A. Lanes 3-5: PJS 130 fragment B. Lane 6: Positive control. Lane 7: Negative control.
  • Two sets of primers used: one to allow insertion into backbone by ligation, and one to allow insertion by Gibson assembly.
  • Cycle settings:
  • Melting - 98 &degC - 10 seconds
  • Annealing - 58 &degC - 30 seconds
  • Elongation - 72 &degC - 100 seconds
  • Products run on gel. Fragments produced of correct size, however they overlapped with the primer dimer band due to the small size of the fragments.
  • DNA extracted and purified.

PCR of Mg2+ riboswitch construct vector


  • Separate reactions for
  • Cycle settings:
  • Melting - 98 &degC - 10 seconds
  • Annealing - 58 &degC - 30 seconds
  • Elongation - 72 &degC - 100 seconds
  • Products run on gel. No fragments produced. Positive control worked however, so master mix clearly works.
  • Given this PCR has worked in the past, will try to re-run with the same settings and hope for success in the future.