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 |
E9 (X-P) |
2 |
1 |
1 |
Ligations were incubated ON at 16°C.
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