Team:UNIPV-Pavia/Notebook/Week3Oct

From 2009.igem.org

EthanolPVanimation.gif

December 2008
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March 2009
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April 2009
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June 2009
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July 2009
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August 2009
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September 2009
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October 2009
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Week from October 12th, to October 18th, 2009

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October, 12th

  • We repeated the digestion for F2620TOP10(S-P) and B5new2-3(X-P-ClaI).
  • Gel run/cut for the two plasmids: bands were good.
  • Gel extraction for:
    • F2620TOP10(S-P)
    • B5new2-3(X-P-ClaI)
    • B3(X-P)
    • B4(X-P)
    • A19-1(S-P)
  • Unfortunately we had a very low DNA yield for almost all of them: A19-1(S-P) and F2620(S-P) could not be used :'(
  • We decided not to repeat A19-1 extraction (and then the A21 re-cloning) and to assay our A21 construct for ethanol production.
  • F2620 DNA was over, so we inoculated 8 ul of F2620MIT1 glycerol stock in 7 ml of LB + Amp. We incubated this inoculum overnight (37°C, 220 rpm).
  • We inoculated 12 of our assemblies in 3 ml of LB + Amp in order to prepare the DNA to send to iGEM HQ:
    • A1
    • A2
    • A3
    • A4
    • A5
    • A6
    • A7
    • A8pg
    • A9pg
    • A11-3
    • A12-2
    • A15-3
  • We ordered a gas chromatography for a sample of the supernatants (taken the previous day).

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October, 13th

  • Miniprep for the 12 inocula to be sent.
  • Miniprep, cut S-P, gel run/cut/purification for F2620MIT1. This time we had a good yield;)
  • Ligations:
    • B14 = F2620(S-P) + B3(X-P) in pSB1A2
    • B15 = F2620(S-P) + B4(X-P) in pSB1A2
  • We incubated them at 16°C overnight.

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October, 14th

  • We transformed the two ligations (after 1:20 dilution) and incubated the plated bacteria overnight (37°C).
  • We inoculated all the remaining assemblies (12) in order to send purified DNA to iGEM HQ (3 ml of LB + Amp).
  • We inoculated 8 ul of B5new2-3, F2620TOP10 and B0015 glycerol stocks in 8 ml of LB + Amp + 2% glucose. We incubated them at 37°C, 220 rpm in the morning.
  • Team meeting

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October, 15th

  • We diluted 25 ul of A14S-3 in 3 ml of LB + Amp and in 3 ml of LB + Amp + 1mM IPTG. We incubated the two cultures at 37°C, 220 rpm for 6 hours. Then we measured OD600 and fluorescence, but unfortunately their GFP levels were the same.
  • Miniprep for the remaining BioBricks.
  • pH measurement for B5new2-3, F2620TOP10 and B0015 24 hour cultures. We diluted 1:100 these three cultures in LB + Amp + 10% glucose and aliquoted them in the microplate reader (anaerobic).
  • We inoculated 4 colonies of B14 plate and 3 colonies of B15 plate in 5 ml of LB + Amp and incubated them at 37°C, 220 rpm overnight. Tomorrow they will be screened!
  • We received gas chromatography results.

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October, 16th

  • Cloning
  • Fermentation experiment setup
  • Wiki updating


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October, 17th

  • Fermentation experiment
  • Wiki updating

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October, 18th

  • Fermentation experiment
  • Wiki updating

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