Team:UNIPV-Pavia/Notebook/Week3Oct

 = Week from October 12th, to October 18th, 2009 =

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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