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 |
| <partinfo>BBa_I13521</partinfo> |
Insert |
12.5 |
8 |
1 EcoRl |
1 Pstl |
2.5 |
25 |
| <partinfo>BBa_R0040</partinfo>-<partinfo>BBa_J61002</partinfo> |
Vector |
20.5 |
0 |
1 EcoRl |
1 Pstl |
2.5 |
25 |
Reactions were incubated at 37°C for three hours; meanwhile two medium-size agarose gels were prepared according to protocols. Moreover BBa_I13521 plasmid purification and quantification were carried out:
In the afternoon gel electrophoresis was performed.
| Plasmid |
DNA (ng/μl) |
| <partinfo>BBa_I13521</partinfo> |
37.3 |
2 righe sopra inserire l' immagine
As shown in figure, all clones were positive (apart from E13 run where we inexplicably didn't see any band), so we cut and purified the bands of interest. Cells harbouring E13 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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