Team:UNIPV-Pavia/Notebook/Week17

Week 17: 09/8/08 - 09/12/08
09/8/08
 * We prepared 0.5 l of LB + Amp for liquid cultures.


 * We infected 9 ml of LB + Amp with 30 µl of Lig.30(3), Lig.31, Lig.22, Lig.13 and two falcon tubes for Lig.a.


 * We received 3OC6HSL from Sigma! we resuspended it in ddH2O, we prepared some stocks (2 mM) and stored them at -20°C.

09/9/08
 * Glycerol stocks/miniprep for Lig.30(3), Lig.31, Lig.22 and Lig.13.


 * Plasmid digestion for:
 * Lig.30 (S-P)
 * Lig.31 (S-P)
 * Lig.30 (X-P)
 * Lig.22 (X-P)
 * 13 (X-P)


 * Run/gel extraction.


 * Ligations:
 * Lig.31-Lig.30 (="Lig.34")
 * Lig.31-Lig.22 (="Lig.35")
 * Lig.30-Lig.13 (="Lig.T5" for green fluorescence test)


 * We induced one of the two Lig.a overnight culture with 3OC6HSL 1 µM. We incubated the two cultures for 1 hour and then watched TRITC channel at microscope. (We didn't synchronize the two cultures, but performed a qualitative test for luxR mutated protein integrity evaluation).


 * Fluorescence test results: HSL induced Lig.a show RFP expression, while Lig.a without HSL showed a weak red fluorescence. Result pictures are not available at the moment.

09/10/08
 * We transformed/plated ligations. We decided to perform two transformations for each ligation:
 * one normal transformation (1 µl of ligation);
 * one diluted ligation (1:10)
 * We decided to try diluted transformations to have less colonies in the plate and to avoid streaking single colonies plates when colonies are not insulated.

09/11/08
 * Plates grew correctly and diluted transformation showed less colonies.


 * Colony PCR for Lig.34, Lig.35 and Lig.T5 (4 colonies for normal transformation plates and 4 colonies for diluted transformation plates).


 * Gel results (gel picture not available, we're sorry...!):
 * Lig.34 (1st,3rd,4th colonies for normal transformation plate; 1st,2nd colonies for diluted transformation plate)
 * Lig.35 (3rd,4th colonies for normal transformation plate; 1st,4th colonies for diluted transformation plate) (this time there were not unexpected contaminants!)
 * Lig.T5 (1st colony)


 * Comments: we chose MANY colonies because Lig.34 and Lig.35 are our final assemblies and we want to be sure that the parts were correct. We decided to extract plasmids from all these colonies and to sequence all of them.


 * We incubated the chosen colonies at 37°C, 220 rpm overnight.

09/12/08
 * Glycerol stocks/miniprep for our ten overnight cultures.


 * We sent:
 * Lig.34-1
 * Lig.34-3
 * Lig.34-4
 * Lig.34dil-1
 * Lig.34dil-2
 * Lig.35-3
 * Lig.35-4
 * Lig.35dil-1
 * Lig.35dil-4
 * to Primm for sequencing.