Team:UNIPV-Pavia/Notebook/Week19

Week 19: 09/22/08 - 09/26/08
09/22/08
 * Sequencing results:
 * Lig.30 - we had another mutation in luxR coding sequence...no comments...(position 349 of C0062, G->C, aminoacid changes...)
 * Lig.34-1 - OK
 * Lig.34-3 - OK
 * Lig.34-4 - OK
 * Lig.34dil-1 - OK
 * Lig.34dil-2 - OK
 * Lig.35-3 - OK
 * Lig.35-4 - OK
 * Lig.35dil-1 - OK
 * Lig.35dil-4 - OK


 * Comments: Lig.34 and Lig.35 sequences were correct, but we have to notice that they are long parts, so their sequencing could not be extended to all the nucleotides. In particular, the mutated nucleotide of Lig.30 was not included in Lig.34 and Lig.35 sequencing. For this reason, we expect to find in Lig.34 the same G->C mutation as Lig.30.


 * We decided to test qualitatively and quantitatively the mutated construct:
 * T5 (R0051-B0030-C0062-B1006-R0062-B0030-E0040-B1006, that carries the G->C mutation)
 * So, we infected 9 ml of LB + Amp with 30 ul of T5 glycerol stock. We incubated the culture at 37°C, 220 rpm overnight.

09/23/08
 * We performed the experiment described in TEST 9 and quantitativeTEST 1 (The Project section, Experiments).


 * We infected 9 ml of LB + Amp with 30 ul of Lig.31 and Lig.36 glycerol stocks. We incubated the cultures at 37°C, 220 rpm overnight.

09/24/08
 * Data processing for 09/23/08 experiment.


 * Glycerol stocks/miniprep for Lig.31 and Lig.36.


 * We sent purified plasmids to Primm for sequencing.

09/24/08
 * We infected 9 ml of LB + Amp with 30 ul of:
 * Lig.34-1
 * T5 (R0051-B0030-C0062-B1006-R0062-B0030-E0040-B1006)
 * Lig.15
 * glycerol stocks. We incubated the three cultures at 37°C, 220 rpm overnight.


 * Using these parts we are going to perform our last ligations...cross the fingers!:)

09/25/08
 * Glycerol stocks/miniprep for Lig.34-1, T5 and Lig.15.


 * Plasmid digestions:
 * Lig.34-1 (S-P)
 * T5 (S-P)
 * Lig.15 (X-P)


 * Gel run/cut. Gel extraction for the three parts.


 * We put purified and digested parts at -20°C. Next week we will perform two ligations!