Team:UNIPV-Pavia/Notebook/Week8

Week 8: 07/7/08 - 07/12/08
07/7/08
 * Colony PCR (5 colonies for each plate) for:
 * BBa_J23100-BBa_B0030-BBa_C0012 (for the second time, we hoped to find true positive colonies)
 * BBa_J23100-BBa_B0030-BBa_I15010 (for the second time, we hoped to find true positive colonies)
 * BBa_B0030-BBa_E0040-BBa_B1006
 * BBa_B0030-BBa_C0051-BBa_B0030
 * BBa_B0030-BBa_E1010-BBa_B1006


 * Gel results:
 * No true positives for BBa_J23100-BBa_B0030-BBa_C0012
 * No true positives for BBa_J23100-BBa_B0030-BBa_I15010
 * Non pure true positives for BBa_B0030-BBa_E0040-BBa_B1006
 * Pure true positives for BBa_B0030-BBa_C0051-BBa_B0030
 * Non pure true positives for BBa_B0030-BBa_E1010-BBa_B1006


 * We chose to keep the first colony for all the 3 working ligation plates.


 * NOTE: BBa_B0030-BBa_E0040-BBa_B1006 and BBa_B0030-BBa_E1010-BBa_B1006 are final parts; we decided to sequence these 2 parts even if gel showed a weak contamination: sequencing results will tell us if there are false positive plasmids in those colonies or if the contamination was only a PCR contamination.


 * We infected 9 ml LB + suitable antibiotic with 30 µl of these glycerol stocks:


 * We incubated the 8 cultures at 37°C, 220 rpm overnight.


 * Tomorrow we will be ready to perform NINE LIGATIONS...(@_@!)

07/8/08
 * We received sequencing results for BBa_B0030-BBa_C0078: sequence showed an extra DNA fragment between BBa_C0078 and Suffix...We decided to ignore it because there was a stop codon before that fragment.


 * Glycerol stocks for:


 * Miniprep for these parts.


 * Plasmid digestion for:


 * Gel run/cut/gel extraction for:


 * DNA precipitation with sodium acetate for:


 * (We already had BBa_I15009(X-P) and BBa_B1006(E-X))


 * Ligations:
 * 1) BBa_B0030-BBa_I15009
 * 2) BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006
 * 3) BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006
 * 4) BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079
 * 5) BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062
 * 6) BBa_J23100-BBa_B0030-BBa_C0012 (again)
 * 7) BBa_J23100-BBa_B0030-BBa_I15010 (again)
 * 8) BBa_J23100-BBa_B0030-BBa_E0040-BBa_B1006 (to test the part)
 * 9) BBa_J23100-BBa_B0030-BBa_E1010-BBa_B1006 (to test the part)


 * We incubated ligations at 16°C overnight.


 * We infected 9 ml LB + Amp with 30 µl of BBa_B1006 and BBa_B0030-BBa_C0078 glycerol stocks. We incubated the 2 cultures at 37°C, 220 rpm ovenight.

07/9/08
 * We transformed the 9 ligations (2 µl) and plated transformed bacteria. We incubated plates at 37°C ovenight.


 * Glycerol stocks for BBa_B1006 and BBa_B0030-BBa_C0078.


 * Miniprep for BBa_B1006 and BBa_B0030-BBa_C0078.


 * Plasmid digestion:


 * Gel run/cut/gel extraction for BBa_B0030-BBa_C0078 (E-S).


 * DNA precipitation with sodium acetate for BBa_B1006 (E-X).


 * Ligation: BBa_B0030-BBa_C0078-BBa_B1006


 * We incubated ligation at 16°C overnight.

07/10/08
 * We transformed BBa_B0030-BBa_C0078-BBa_B1006 ligation (2 µl) and plated transformed bacteria. We incubated the plate at 37°C ovenight.


 * All the ligation plates showed colonies! There were carpets, but we could pick up some single colonies for colony PCR.


 * Colony PCR for:
 * 1) BBa_B0030-BBa_I15009
 * 2) BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006
 * 3) BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006
 * 4) BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079
 * 5) BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062
 * 6) BBa_J23100-BBa_B0030-BBa_C0012
 * 7) BBa_J23100-BBa_B0030-BBa_I15010


 * Gel results:
 * 1) BBa_B0030-BBa_I15009 1st colony was chosen.
 * 2) BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006 5th colony was chosen.
 * 3) BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 2nd colony was chosen.
 * 4) BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079 4th colony was chosen.
 * 5) BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062 3rd colont was chosen.
 * 6) BBa_J23100-BBa_B0030-BBa_C0012 did not show true positive colonies.
 * 7) BBa_J23100-BBa_B0030-BBa_I15010 did not show true positive colonies.


 * We streaked BBa_J23100-BBa_B0030-BBa_E0040-BBa_B1006 and BBa_J23100-BBa_B0030-BBa_E1010-BBa_B1006 with a top to test the parts (NOTE: we could see some red colonies on BBa_J23100-BBa_B0030-BBa_E1010-BBa_B1006 plate!these colonies correspond to bacteria with correctly ligated plasmid, while normal color colonies correspond to bacteria with BBa_B0030-BBa_E1010-BBa_B1006 plasmid, without constitutive promoter).
 * We infected 100 µl LB + Amp with the tips and incubated the culture at 37°C, 220 rpm for 2 hours.
 * Then we watched green, blue and red fluorescence channels at microscope (50 µl of each culture):


 * LB preparation: 0.5 l LB + Amp for plates.


 * BBa_R0010 resuspension from Registry of Standard Parts.


 * We transformed (4 µl) BBa_R0010 and plated transformed bacteria. We incubated BBa_R0010 plate at 37°C overnight.


 * We infected 9 ml LB + suitable antibiotic with 30 µl of these glycerol stocks:


 * We also picked up one colony from BBa_I15010 plate with a tip and infected 9 ml LB + Kan. We incubated the 8 cultures at 37°C, 220 rpm overnight.

07/11/08
 * BBa_R0010 plate showed 2 colonies. We picked up one colony and infected 9 ml LB + Amp to grow an overnight culture.


 * BBa_B0030-BBa_C0078-BBa_B1006 plate showed many colonies. We used 7 colonies to perform colony PCR.


 * Gel results: all the 7 colonies contained ligated plasmid; we chose the 5th colony to grow a 9 ml overnigth culture because 5th it showed no impurities.


 * Glycerol stocks for:


 * Miniprep for these 8 parts.


 * We sent BBa_I15010 and BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062 (3) to Primm for sequencing.


 * Plasmid digestion for:


 * Gel run/cut/gel extraction for all these parts.


 * (We already had BBa_B1006 (E-X) and BBa_R0062 (E-X))


 * Ligation:
 * 1) BBa_J23100-BBa_B0030 (S-P) -BBa_C0012 (X-P) (1:2 ratio instead of 1:6)
 * 2) BBa_B1006 (E-P) + BBa_J23100-BBa_B0030 (E-S, 1:4) -BBa_C0012 (X-P, 1:2) (we decided to try this double ligation)
 * 3) BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040
 * 4) BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062
 * 5) BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006
 * 6) BBa_B0030-BBa_I15009-BBa_B1006


 * We incubated ligations at 16°C overnight.


 * We infected 9 ml LB + Amp with 30 µl of BBa_R0079 glycerol stock.

07/12/08
 * Glycerol stocks for:


 * Miniprep for these 3 parts.


 * We put these 3 purified plasmids at -20°C.


 * We also put the 6 ovenight ligations at -20°C. Next week they will be transformed!