July, 18th
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) |
| E9-2 |
Insert |
11.5 |
9 |
1 Xbal |
1 Pstl |
2.5 |
25 |
| E10-1 |
Insert |
10 |
10.5 |
1 Xbal |
1 Pstl |
2.5 |
25 |
| E11-1 |
Insert |
4 |
16.5 |
1 Xbal |
1 Pstl |
2.5 |
25 |
| E12-1 |
Insert |
10 |
10.5 |
1 Xbal |
1 PstI |
2.5 |
25 |
| E13-1 |
??Insert?? |
6 |
14.5 |
1 EcoRl |
1 Pstl |
2.5 |
25 |
| E16-1 |
??Insert?? |
4 |
16.5 |
1 EcoRl |
1 Pstl |
2.5 |
25 |
| E21-1 |
??Insert?? |
7.5 |
13 |
1 EcoRl |
1 Xbal |
2.5 |
25 |
| E22-2 |
??Insert?? |
10 |
10.5 |
1 EcoRl |
1 Pstl |
2.5 |
25 |
| I13521 |
Insert |
12.5 |
8 |
1 EcoRl |
1 Pstl |
2.5 |
25 |
| R0040-J61002 |
Vector |
20.5 |
0 |
1 EcoRl |
1 Pstl |
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.
2 righe sopra inserire l' immagine
As shown, in figure all clones were positive (apart from <partinfo>BBa_I13501</partinfo> run where we inexplicably didn't see any band), so we cut and purified the bands of interest. Cells harbouring <partinfo>BBa_I13501</partinfo> were again inoculated in 5 ml LB + Amp.
After gel extraction, cut DNA was quantified:
| Plasmid |
DNA (ng/μl) |
| <partinfo>BBa_C0060</partinfo> (E-S) |
3.9 |
| <partinfo>BBa_C0061</partinfo> (X-P) |
4.1 |
| <partinfo>BBa_K081022</partinfo> (E-P) |
4.4 |
| <partinfo>BBa_B0030</partinfo> (S-P) |
10.3 |
| <partinfo>BBa_B0031</partinfo> (S-P) |
11.8 |
| <partinfo>BBa_B0032</partinfo> (S-P) |
10.1 |
| <partinfo>BBa_B0015</partinfo> (E-X) |
12 |
| <partinfo>pSB4C5</partinfo> (E-P) |
10.7 |
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) |
| E1 |
<partinfo>BBa_B0015</partinfo> (E-X) |
1.5 |
<partinfo>BBa_C0060</partinfo> (E-S) |
6.5 |
1 |
1 |
| E2 |
<partinfo>BBa_B0030</partinfo> (S-P) |
1.5 |
<partinfo>BBa_C0061</partinfo> (X-P) |
6.5 |
1 |
1 |
| E3 |
<partinfo>BBa_B0031</partinfo> (S-P) |
1.5 |
<partinfo>BBa_C0061</partinfo> (X-P) |
6.5 |
1 |
1 |
| E4 |
<partinfo>BBa_B0032</partinfo> (S-P) |
1.5 |
<partinfo>BBa_C0061</partinfo> (X-P) |
6.5 |
1 |
1 |
| E8 |
<partinfo>pSB4C5</partinfo> (E-P) |
1.5 |
<partinfo>BBa_K081022</partinfo> (E-P) |
6.5 |
1 |
1 |
Ligations were incubated ON at 16°C.
July, 19th
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