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 it is possible to recognize only a band at approximately 2800 bp, corresponding to the undigested plasmid), so we cut and purified the bands of interest. Cells harbouring E13 were again inoculated in 5 ml LB + Amp.
E13 was newly then digested with restriction endonucleases:
| Plasmid |
Kind |
DNA (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume (μl) |
| <partinfo>BBa_I13521</partinfo> |
Insert |
1 |
19.5 |
1 EcoRl |
1 Pstl |
2.5 |
25 |
After gel extraction, cut DNA was quantified:
| Plasmid |
DNA (ng/μl) |
| E9 (X-P) |
5.9 |
| E10 (X-P) |
4.2 |
| E11 (X-P) |
5.2 |
| E12 (X-P) |
5.2 |
| E16 (E-P) |
6.4 |
| E21 (E-P) |
5.8 |
| E22 (E-P) |
6.5 |
| E23 (E-P) |
4.6 |
| <partinfo>BBa_I13521</partinfo> |
6.1 |
| <partinfo>BBa_R0040</partinfo>-<partinfo>BBa_J61002</partinfo> |
2.1 |
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) |
| E24 |
<partinfo>BBa_R0040</partinfo> (S-P) |
1.5 |
E9 (X-P) |
6.5 |
1 |
1 |
| E25 |
<partinfo>BBa_R0040</partinfo> (S-P) |
1.5 |
E10 (X-P) |
6.5 |
1 |
1 |
| E26 |
<partinfo>BBa_R0040</partinfo> (S-P) |
1.5 |
E11 (X-P) |
6.5 |
1 |
1 |
| E27 |
<partinfo>BBa_R0040</partinfo> (S-P) |
1.5 |
E12 (X-P) |
6.5 |
1 |
1 |
| E31 |
<partinfo>pSB4C5</partinfo> (E-P) |
2.5 |
E16 (E-P) |
5.5 |
1 |
1 |
| E32 |
<partinfo>pSB4C5</partinfo> (E-P) |
2 |
E21 (E-P) |
6 |
1 |
1 |
| E33 |
<partinfo>pSB4C5</partinfo> (E-P) |
2 |
E22 (E-P) |
6 |
1 |
1 |
| E34 |
<partinfo>pSB4C5</partinfo> (E-P) |
6.5 |
E23 (E-P) |
1.5 |
1 |
1 |
| E35 |
<partinfo>pSB4C5</partinfo> (E-P) |
2 |
<partinfo>BBa_I13521</partinfo>(E-P) |
6.5 |
1 |
1 |
| E36 |
<partinfo>pSB4C5</partinfo> (E-P) |
1 |
<partinfo>BBa_R0040</partinfo>-<partinfo>BBa_J61002</partinfo>(E-P) |
7 |
1 |
1 |
Ligations were incubated ON at 16°C.
July, 19th
E13 was newly digested for the subsequent gel extraction.
| Plasmid |
Kind |
DNA (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume (μl) |
| E13 |
Insert |
6 |
14.5 |
1 Xbal |
1 Pstl |
2.5 |
25 |
After electrophoresis, E13 insert was succesfully identified and gel extracted (not shown). Meanwhile, sufficient amount of buffer 1 and buffer 2 for MGZ1 competent cell preparation protocol were prepared. E13 DNA was then quantified:
| Plasmid |
DNA (ng/μl) |
| E13 (X-P) |
43 |
After E13 digestion (E-P), its ligation was performed in a final volume of 10 μl:
| Ligation Name |
Vector |
Vector volume (μl) |
Insert |
Insert volume (μl) |
Buffer (μl) |
T4 Ligase (μl) |
| E28 |
<partinfo>pSB4C5</partinfo> (E-P) |
6 |
E13 (X-P) |
2 |
1 |
1 |
Ligations were incubated ON at 16°C.
250 ml of M9 were prepared, according to protocols.
July, 20th
E24, E25, E26, E27 and were transformed in 100 μl of TOP10 competent cells according to protocols. Moreover E28, E31, E32, E33, E34, E35, E36 and <partinfo>B0032</partinfo> (transformation efficiency positive control) were transformed in 100 μl of MGZ1 competent cells according to protocols. Plates were incubated ON at 37°C.
500 ml of LB with chloramphenicol 12.5 were prepared.
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