Team:UNIPV-Pavia/Notebook/Week7

Week 7: 06/30/08 - 06/5/08
06/30/08
 * We received sequencing results for BBa_B0030-BBa_C0061 (4th colony and 7th colony): sequences were correct! We decided to keep the 4th colony.


 * Colony PCR for BBa_J23100-BBa_E0240 and BBa_B0030-BBa_C0061: 5 colonies for every plate. Gel showed many working colonies: we chose first colonies for the two ligations.


 * We infected 9 ml LB + Amp with 30 µl of:


 * Tomorrow we will be ready to perform 5 ligations!

07/1/08
 * Glycerol stocks for:


 * Miniprep for all these parts.


 * We sent BBa_J23100-BBa_E0240 (1) and BBa_B0030-BBa_C0078 (1) to Primm for sequencing.


 * We performed digestion for:


 * Gel run/cut for:


 * Gel extraction for these 5 parts.


 * DNA precipitation with sodium acetate for:


 * Ligation:
 * BBa_J23100-BBa_B0030-BBa_C0012
 * BBa_J23100-BBa_B0030-BBa_C0040
 * BBa_J23100-BBa_B0030-BBa_I15010
 * BBa_R0051-BBa_B0030-BBa_C0062
 * BBa_B0030-BBa_C0061-BBa_B1006


 * We incubated ligation reaction at 16°C overnight.

07/2/08
 * We transformed ligations (5 µl) and plated transformed bacteria.


 * We also transformed BBa_B1006(E-X), BBa_J23100-BBa_B0030(S-P) and BBa_R0051-BBa_B0030 (1 µl) and plated transformed bacteria to estimate background noise.


 * We received sequencing results for:
 * BBa_B0030-BBa_E0040
 * BBa_B0030-BBa_C0051
 * BBa_B0030-BBa_E1010
 * All the sequences were correct!


 * We infected 9 ml LB + Amp with 30 µl of:
 * glycerol stocks.

07/3/08
 * All the ligation plates showed carpets, while negative control plates showed a weak background noise.


 * Single colonies plates for ligation plates.


 * Glycerol stocks for:


 * Miniprep for these parts.


 * We performed digestion:


 * Gel run/cut for:


 * Gel extraction.


 * DNA precipitation with sodium acetate for:

Ligation:
 * BBa_B0030-BBa_E0040-BBa_B1006
 * BBa_B0030-BBa_C0051-BBa_B0030
 * BBa_B0030-BBa_E1010-BBa_B1006


 * We incubated ligation reactions at 16°C overnight.

07/4/08
 * We transformed the 3 ligations (2 µl) and plated transformed bacteria.


 * Colony PCR for single colonies plates (3 colonies for each plate):


 * Electrophoresis for PCR result: unfortunately, BBa_J23100-BBa_B0030-BBa_C0012 and BBa_J23100-BBa_B0030-BBa_I15010 did not show any true positive colony. We decided to re-perform colony PCR for these two parts next week. We chose to keep the first colonies for the other 3 ligations to grow 9 ml cultures overnight.

07/5/08
 * All the ligation plates showed colonies! Next week we will perform colony PCR to find true positive colonies.


 * Glycerol stocks and Miniprep for: BBa_J23100-BBa_B0030-BBa_C0040 (1), BBa_R0051-BBa_B0030-BBa_C0062 (1), BBa_B0030-BBa_C0061-BBa_B1006 (1).