July, 11th
| Plasmid |
Kind |
DNA (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume (μl) |
| E1-2 |
|
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E2-2 |
|
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E3-1 |
|
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E4-2 |
|
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E5-2 |
|
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E6-1 |
|
11 |
9.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E7-2 |
|
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| E8-3 |
|
20.5 |
0 |
1 SpeI |
1 PstI |
2.5 |
25 |
| <partinfo>BBa_R0040</partinfo> |
|
14.5 |
6 |
1 SpeI |
1 PstI |
2.5 |
25 |
| <partinfo>BBa_C0261</partinfo> |
|
18 |
2.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| <partinfo>BBa_I13507</partinfo> |
|
10 |
10.5 |
1 XbaI |
1 PstI |
2.5 |
25 |
| <partinfo>BBa_B0034</partinfo> |
|
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.
link alle 2 img dei gel con ling wiki
As shown, in figure all clones were positive, so we cut and purified the bands of interest.
After gel extraction, cut DNA was quantified:
| Plasmid |
DNA (ng/μl) |
| E1 (X-P) |
4.2 |
| E2 (X-P) |
3.8 |
| E3 (X-P) |
4.1 |
| E4 (X-P) |
4.2 |
| E5 (X-P) |
5.6 |
| E6 (X-P) |
7.8 |
| E7 (X-P) |
6.4 |
| E8 (S-P) |
3.4 |
| <partinfo>BBa_C0261</partinfo> (X-P) |
4.8 |
| <partinfo>BBa_R0040</partinfo> (S-P) |
7.9 |
| <partinfo>BBa_B0034</partinfo> (S-P) |
11.0 |
| <partinfo>BBa_I13507</partinfo> (X-P) |
3.9 |
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 |
<partinfo>BBa_B0030</partinfo> (S-P) |
1 |
E1 (X-P) |
7 |
1 |
1 |
| E10 |
<partinfo>BBa_B0031</partinfo> (S-P) |
1 |
E1 (X-P) |
7 |
1 |
1 |
| E11 |
<partinfo>BBa_B0032</partinfo> (S-P) |
1 |
E1 (X-P) |
7 |
1 |
1 |
| E12 |
<partinfo>BBa_B0034</partinfo> (S-P) |
1 |
E1 (X-P) |
7 |
1 |
1 |
| E13 |
<partinfo>BBa_R0040</partinfo> (S-P) |
1.5 |
E2 (X-P) |
6.5 |
1 |
1 |
| E14 |
<partinfo>BBa_R0040</partinfo> (S-P) |
1.5 |
E3 (X-P) |
6.5 |
1 |
1 |
| E15 |
<partinfo>BBa_R0040</partinfo> (S-P) |
1.5 |
E4 (X-P) |
6.5 |
1 |
1 |
| E16 |
<partinfo>BBa_R0040</partinfo> (S-P) |
2 |
<partinfo>BBa_C0261</partinfo> (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 |
<partinfo>BBa_I13507</partinfo> (X-P) |
3 |
1 |
1 |
| E21 |
<partinfo>BBa_R0040</partinfo> (S-P) |
2 |
E5 (X-P) |
6 |
1 |
1 |
| E22 |
<partinfo>BBa_R0040</partinfo> (S-P) |
2.5 |
E6 (X-P) |
5.5 |
1 |
1 |
| E23 |
<partinfo>BBa_R0040</partinfo> (S-P) |
2 |
E7 (X-P) |
6 |
1 |
1 |
Ligations were incubated ON at 16°C.
July, 12th
E9, E10, E11, E12, E13, E14, E15, E16, E17, E18, 19, E20, E21, E22, E23, and E8 were transformed in 100 μl of TOP10 competent cells according to protocols. Plates were incubated ON at 37°C.
July, 13th
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