Team:Cambridge/Lab book/Week 7

From 2012.igem.org

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===Monday (06/08/12)====
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===Monday (06/08/12)===
'''[[Team:Cambridge/Protocols/PCRProtocol|PCR of Magnesium riboswitch vector fragment B and Magnesium promoter]]'''
'''[[Team:Cambridge/Protocols/PCRProtocol|PCR of Magnesium riboswitch vector fragment B and Magnesium promoter]]'''
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*Positive control also failed, although this has ceased to work for several days, potentially due to DNA degradation.
*Positive control also failed, although this has ceased to work for several days, potentially due to DNA degradation.
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===Tuesday (07/08/12)====
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===Tuesday (07/08/12)===
'''[[Team:Cambridge/Protocols/Electrocompetentcells|Production of electrocompetent e.coli]]'''
'''[[Team:Cambridge/Protocols/Electrocompetentcells|Production of electrocompetent e.coli]]'''

Revision as of 09:14, 8 August 2012

Week: 3 4 5 6 7

Contents

Monday (06/08/12)

PCR of Magnesium riboswitch vector fragment B and Magnesium promoter


Riboswitch and promoter gel. Lanes 2+3: Fragment B without 8 codon substitution, lanes 4+5: Fragment B with 8 codon substitution, lanes 6+7: magnesium sensitive promoter, lane 8: +ve control
  • Normal PCR settings used, annealing temperature 57 °C, elongation step 90s long.
  • Lane 5 accidentally loaded with a DNA ladder instead of loading dye.
  • Expected fragment sizes:
  • Lane 2-5: 3kbp
  • Lane 6-7: 300bp
  • After electrophoresis, found vector products had, for the most part, worked. Promoter elements were not amplified - no band of the expected size was observed.
  • Positive control also failed, although this has ceased to work for several days, potentially due to DNA degradation.

Tuesday (07/08/12)

Production of electrocompetent e.coli


Gibson assembly of magnesium riboswitch and fluorescent construct


  • NAD+ added to isothermal buffer*5 mix
  • Gel slices from yesterday (of vector fragment B) purified.
  • DNA added as follows:
  • Without 8 codon substitution:
  • Reaction 1: Tube 2 (07/08/12) (fragment B), Tube 1 (05/08/12) (fragment A), Tube 28 (29/08/12) (riboswitch DNA).
  • Reaction 2: Tube 3 (07/08/12) (fragment B), Tube 2 (05/08/12) (fragment A), Tube 29 (29/08/12) (riboswitch DNA).
  • With 8 codon substitution:
  • Reaction 3: Tube 4 (07/08/12) (fragment B), Tube 1 (05/08/12) (fragment A), Tube 2 (18/07/12) (riboswitch DNA).

Wednesday (08/08/12)

Thursday

Friday