Team:UNIPV-Pavia/Notebook/Week10

Week 10: 07/21/08 - 07/25/08
07/21/08
 * Colony PCR for B0030-C0078-B1006-R0079: 6 colonies from single colonies plate.


 * Gel results: all the 6 picked colonies were true positive! we chose the 5th colony to grow a 9 ml overnight culture.


 * We also infected 9 ml LB + Amp with 30 µl of J23100-B0030-C0040-B1006-R0040 and B0030-C0061 glycerol stocks to grow two overnight cultures.


 * Ligation:
 * J23100-B0030-C0012-B1006 (1:2 ratio, 40 ng of vector)
 * B0030-I15009-B1006-R0082 (30 ng of vector)
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006


 * We incubated ligations at 16°C overnight.

07/22/08
 * Transformation for the 4 overnight ligations (1 µl). We plated transformed bacteria and incubated plates at 37°C overnight.


 * Glycerol stocks/miniprep for:


 * We sent J23100-B0030-C0040-B1006-R0040 and B0030-C0078-B1006-R0079 purified plasmids to Primm for sequencing.


 * Plasmid digestion for:


 * Gel run/cut/gel extraction.

07/23/08
 * Ligation plates showed colonies!We put ligation plates at +4°C.


 * Ligation for B0030-C0061-B0030-C0078-B1006-R0079.


 * We incubated ligation at 16°C overnight.

07/24/08
 * Transformation (1 µl) for B0030-C0061-B0030-C0078-B1006-R0079 ligation. We plated transformed bacteria and incubated plate at 37°C overnight.


 * Colony PCR for:
 * J23100-B0030-C0012-B1006
 * B0030-I15009-B1006-R0082
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006


 * (We performed one PCR for J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082 with 2 min elongation and another PCR for the remaining two ligations with 3 min elongation).


 * Electrophoresis (1.2% agarose) for J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082:


 * Electrophoresis (1% agarose) for B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006:


 * Gel results:
 * J23100-B0030-C0012-B1006 1st colony
 * B0030-I15009-B1006-R0082 1st colony
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 1st and 3rd colony
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 1st and 4th colony


 * We decided to keep two colonies for B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 ligations because they are important parts for our project and we wanted to be sure they were correct.


 * The fifth colony of B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 showed an unexpected length...(@_@?) We decided to ignore it. Sequencing results will tell us if we made mistakes in assemblies.


 * We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures.

07/25/08
 * Glycerol stocks/miniprep for:
 * J23100-B0030-C0012-B1006 (1)
 * B0030-I15009-B1006-R0082 (1)
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1)
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (3)
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (1)
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (4)


 * We sent these purified plasmids:
 * B0030-I15009-B1006-R0082 (1)
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1)
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (3)
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (1)
 * B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (4)


 * to Primm for sequencing.


 * We put B0030-C0061-B0030-C0078-B1006-R0079 plate at +4°C.