Team:UNIPV-Pavia/Notebook/Week15

Week 15: 08/25/08 - 08/29/08
08/25/08
 * Plasmid digestion for:
 * R0051 (S-P)
 * R0040 (S-P)
 * B0030-C0061-B1006-R0062-B0030-E0040-B1006 (E-X) (=Lig.b (E-X))
 * B1006 (E-X)
 * Lig.12 (E-S)


 * Run/gel extraction.


 * Ligations:
 * BBa_R0051 (S-P) - BBa_E0240 (X-P) (for promoter test)
 * BBa_R0040 (S-P) - BBa_E0240 (X-P) (for promoter test)
 * Lig.12 (E-S) - B0030-C0061-B1006-R0062-B0030-E0040-B1006 (E-X) (for AND logic gate test)
 * Lig.12 (E-S) - BBa_B1006 (E-X) (to re-perform mutated assemblies)
 * We incubated ligations at 16°C overnight.


 * We ordered 3OC6HSL (Sigma).

08/26/08
 * We transformed/plated ligations.

08/27/08
 * Plates grew correctly. We checked colony fluorescence of three plates under UV rays:
 * R0051-E0240 glowed
 * R0040-E0240 glowed
 * Lig.12-Lig.b (R0051-B0030-C0062-B0030-C0061-B1006-R0062-B0030-E0040-B1006) glowed


 * We picked up fluorescent colonies from these three plates to infect three 15 ml falcon tubes containing 1 ml of LB + Amp. We let the culture grow at 37°C, 220 rpm for 3 hours, then we prepared three 50 ul samples and watch them at microscope. Results are shown in The Project section (Experiments).


 * Colony PCR for 5 colonies of Lig.12-B1006 (called Lig.22).


 * Gel results: OK! we chose 1st colony to grow a 9 ml overnight culture.

08/28/08
 * Glycerol stocks/miniprep for Lig.22.


 * We sent purified plasmids to Primm for sequencing.


 * We infected 9 ml of LB + Amp with 30 µl of R0062 glycerol stock.

08/29/08
 * Glycerol stock/miniprep for R0062.