July, 11th
Digestions of previously purified plasmids were performed for ligations:
| Plasmid |
Kind |
DNA (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume (μl) |
| E1-2 |
Insert |
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E2-2 |
Insert |
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E3-1 |
Insert |
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E4-2 |
Insert |
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E5-2 |
Insert |
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E6-1 |
Insert |
11 |
9.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E7-2 |
Insert |
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E8-3 |
Vector |
20.5 |
0 |
1 SpeI |
1 PstI |
2.5 |
25 |
| BBa_R0040 |
Vector |
14.5 |
6 |
1 SpeI |
1 PstI |
2.5 |
25 |
| BBa_C0261 |
Insert |
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| BBa_I13507 |
Insert |
10 |
10.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| BBa_B0034 |
Vector |
10 |
10.5 |
1 SpeI |
1 PstI |
2.5 |
25 |
Reactions were incubated at 37°C for three hours while two medium-size agarose gels were prepared according to protocols.
In the afternoon gel electrophoresis was performed.
Medium size gel
Medium size gel
As shown, in figure all clones were positive, so we cut and purified the bands of interest.
After gel extraction, digested DNA was quantified:
| Part |
DNA (ng/μl) |
| E1 (X-P) |
4.2 |
| E2 (X-P) |
3.8 |
| E3 (X-P) |
4.1 |
| E4 (X-P) |
3.7 |
| E5 (X-P) |
5.6 |
| E6 (X-P) |
7.8 |
| E7 (X-P) |
6.4 |
| E8 (S-P) |
3.4 |
| BBa_C0261 (X-P) |
4.8 |
| BBa_R0040 (S-P) |
7.9 |
| BBa_B0034 (S-P) |
11.0 |
| BBa_I13507 (X-P) |
6.8 |
Then ligations were performed in a final volume of 10 μl:
| Ligation Name |
Vector |
Vector volume (μl) |
Insert |
Insert volume (μl) |
Buffer (μl) |
T4 Ligase (μl) |
| E9 |
BBa_B0030 (S-P) |
1 |
E1 (X-P) |
7 |
1 |
1 |
| E10 |
BBa_B0031 (S-P) |
1 |
E1 (X-P) |
7 |
1 |
1 |
| E11 |
BBa_B0032 (S-P) |
1 |
E1 (X-P) |
7 |
1 |
1 |
| E12 |
BBa_B0034 (S-P) |
1 |
E1 (X-P) |
7 |
1 |
1 |
| E13 |
BBa_R0040 (S-P) |
1.5 |
E2 (X-P) |
6.5 |
1 |
1 |
| E14 |
BBa_R0040 (S-P) |
1.5 |
E3 (X-P) |
6.5 |
1 |
1 |
| E15 |
BBa_R0040 (S-P) |
1.5 |
E4 (X-P) |
6.5 |
1 |
1 |
| E16 |
BBa_R0040 (S-P) |
2 |
BBa_C0261 (X-P) |
6 |
1 |
1 |
| E17 |
E8 (S-P) |
4.5 |
E5 (X-P) |
3.5 |
1 |
1 |
| E18 |
E8 (S-P) |
5 |
E6 (X-P) |
3 |
1 |
1 |
| E19 |
E8 (S-P) |
5 |
E7 (X-P) |
3 |
1 |
1 |
| E20 |
E8 (S-P) |
5 |
BBa_I13507 (X-P) |
3 |
1 |
1 |
| E21 |
BBa_R0040 (S-P) |
2 |
E5 (X-P) |
6 |
1 |
1 |
| E22 |
BBa_R0040 (S-P) |
2.5 |
E6 (X-P) |
5.5 |
1 |
1 |
| E23 |
BBa_R0040 (S-P) |
2 |
E7 (X-P) |
6 |
1 |
1 |
Ligations were incubated ON at 16°C.
July, 12th
E9, E10, E11, E12, E14, E15, E17, E18, E19, E20, E21, E22 and E23 were transformed in 100 μl of TOP10 competent cells according to protocols, while E13 and E16 ligations were transformed in 100 μl of MGZ1 competent cells. Plates were incubated ON at 37°C.
July, 13th
All plates showed a lot of colonies, except for E13, E16 (one colony),while for E14, E15 no colonies were observed (probably the parts gave too high metabolic burden that prevents cells growth or because of low transformation efficiency of MGZ1 strain).
Because of RFP, E21 was red, E22 and E23 a little bit less.
Two colonies (where possible) were picked for each plate.
500 ml of LB with Ampicillin were prepared.
Glycerol stocks were prepared for every culture, except for E11-1, E21-2, E23-2 because they were not sufficiently grown.
July, 14th
Glycerol stocks for E11-1, E21-2 and E23-2 were prepared.
Cultures grew overnight; plasmid purification and quantification were carried out:
| Plasmid |
DNA (ng/μl) |
| E9-1 |
114 |
| E10-1 |
72 |
| E11-1 |
78.5 |
| E12-1 |
151.7 |
| E13-1 |
133 |
| E16-1 |
134.4 |
| E17-1 |
27.8 |
| E18-1 |
25.5 |
A 25 x mix was prepared in order to perform PCR on E9-1, E9-2, E10-1, E10-2, E11-1, E11-2, E12-1, E12-2, E17-1, E17-2, E18-1, E18-2, E19-1, E19-2, E20-1, E20-2, E21-1, E21-2, E22-1, E22-2, E23-1, E23-2:
| H2O (μl) |
Buffer 10x (μl) |
MgCl2 (μl) |
VF2 (BBa_G00100) μl |
VR (BBa_G00101) μl |
dNTPs (μl) |
Taq polymerase (μl) |
| 450 |
62.5 |
25 |
12.5 |
12.5 |
12.5 |
25 |
24 μl were aliquoted in each tube, together with 1 μl of each liquid culture. PCR cycles parameters were set as follows:
- 94°C 30 seconds (denaturing)
- 60°C 1 minute (annealing)
- 72°C 2 minutes (elongation)
35 cycles were performed.
In order to screen the DNA for the only two colonies of E13 and E16, a digestion for each ligation was performed.
| Plasmid |
DNA (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume (μl) |
| E13-1 |
3 |
18.5 |
0.5 EcoRI |
0.5 PstI |
2.5 |
25 |
| E16-1 |
3 |
18.5 |
0.5 EcoRI |
0.5 PstI |
2.5 |
25 |
Reactions were incubated at 37°C for three hours.
BBa_B0032 were transformed in 100 μl of MGZ1 competent cells to test if there was any problem with their transformation efficiency. Plates were incubated ON at 37°C.
Two medium size gels were prepared.
In the afternoon gel electrophoresis was performed.
Medium size gel (20 wells)
Medium size gel (15 wells)
As shown in figure, all clones were positive, except for E9-1,E17-2, E18-1, E18-2, E19-1 which didn't show any DNA band.
E9-2, E17-2, E18-2, E19-2, E20-2, E21-1, E22-2, E23-1 purification was carried out for sequencing.
| Plasmid |
DNA (ng/μl) |
| E9-2 |
66 |
| E17-2 |
28 |
| E18-2 |
26.3 |
| E19-2 |
39.1 |
| E20-2 |
22.1 |
| E21-1 |
143.1 |
| E22-2 |
70.4 |
| E23-1 |
80.9 |
| BBa_I13521 |
79 |
Cells harbouring E9-2, E10-1, E11-1, E12-1, E13, E16, E21-1, E22-2, E23-1 were inoculated in???? 5 ml???? LB + Amp.
July, 15th
All plates showed a lot of colonies, but the plate containing MGZ1 transformed with BBa_B0032 also showed a lot of satellities.
In order to screen the DNA, digestionS for E17-1, E17-2, E18-1, E18-2 ligation were performed.
| Ligation Name |
DNA (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume (μl) |
| E17-1 |
6 |
15.5 |
0.5 EcoRI |
0.5 PstI |
2.5 |
25 |
| E17-2 |
6 |
15.5 |
0.5 EcoRI |
0.5 PstI |
2.5 |
25 |
| E18-1 |
6 |
15.5 |
0.5 EcoRI |
0.5 PstI |
2.5 |
25 |
| E18-2 |
6 |
15.5 |
0.5 EcoRI |
0.5 PstI |
2.5 |
25 |
"
