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 |
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