Team:UNIPV-Pavia/Notebook/Week1Jun

 = Week from June 1st, to June 7th, 2009 =

June, 3rd

 * We received lactose monohydrate, M9 minimal salts and Thiamine Hydrochloride from Sigma.


 * DNA resuspension from iGEM 2009 plates. We resuspended the two parts we needed to re-built the inconsistent BioBrick Q04400, the five commonly used RBSs and a 3OC6-HSL inducible measurement system for GFP test at Tecan F200:


 * Transformation of resuspended DNA (2 ul) in TOP10 E. coli, plated transformed bacteria and incubated the plates overnight at 37°C.


 * We streaked a plate with a 2008 glycerol stock containing E0240 under the control of a constitutive promoter (we call this construct "01") in order to use it in the fluorescence test at Tecan F200 on June 5th.

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June, 4th
Unfortunately, B0034 and B0032 plates also showed a very small amount of unexpected red colonies...(@_@!?) These two parts had been resuspended from iGEM 2009 plate 1, just like Berkeley constitutive promoters, but it is not clear how they could be contaminated...
 * All the overnight plates showed colonies. 01 plate showed green fluorescence under UV rays, as we expected because GFP was expressed constitutively!


 * Anyway, we decided to continue our work and we picked one colony from the following plates:

and infected 1 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 5 and 1/2 hours. Of course, we carefully avoided to pick red colonies from B0034 and B0032 plates:) Sequencing checks will tell us if our parts are correct.


 * Glycerol stocks for all the grown cultures (except for 01, because we already had it).


 * NOTE: we re-picked colonies from J23100, J23101 and J23118 plates because data analysis of experiment 1 at Tecan F200 showed that J23101 promoter seemed stronger than J23100. This is not in accordance to the ranking of the promoters (http://partsregistry.org/Promoters/Catalog/Anderson), so we decided to repeat the test at Tecan F200 with brand new colonies and to store these new glycerol stocks at -80°C.


 * Gel results: the 3 colonies of P0440 all have the plasmid with the correct length of the insert (1078 bp), while there have been problems with R0040 colonies. R0040-1 and R0040-2 show the correct length of the insert (292 bp) with a high weight contaminant and R0040-3 show an unexpected wrong length. Next week we are going to digest P0440 and R0040 to perform their assembly, so we will check the actual plasmid and insert length again.

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June, 5th
Experiment with Tecan F200


 * Download Protocol

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June, 6th
Experiment with Tecan F200


 * Results check

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