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 |
| E17 |
E8 (S-P) |
4.5 |
E5 (X-P) |
3.5 |
1 |
1 |
| E18 |
E8 (S-P) |
5 |
E6 (X-P) |
3 |
1 |
1 |
| E19 |
E8 (S-P) |
5 |
E7 (X-P) |
3 |
1 |
1 |
| E20 |
E8 (S-P) |
5 |
<partinfo>BBa_I13507</partinfo> (X-P) |
3 |
1 |
1 |
| E21 |
<partinfo>BBa_R0040</partinfo> (S-P) |
2 |
E5 (X-P) |
6 |
1 |
1 |
| E22 |
<partinfo>BBa_R0040</partinfo> (S-P) |
2.5 |
E6 (X-P) |
5.5 |
1 |
1 |
| E23 |
<partinfo>BBa_R0040</partinfo> (S-P) |
2 |
E7 (X-P) |
6 |
1 |
1 |
Ligations were incubated ON at 16°C.
July, 12th
E9, E10, E11, E12, E13, E14, E15, E16, E17, E18, 19, E20, E21, E22, E23, and E8 were transformed in 100 μl of TOP10 competent cells according to protocols. Plates were incubated ON at 37°C.
July, 13th
All plates showed a lot of colonies, except for E13, E16 (one colony), E14, E15 (zero colony).
Because of RFP, E21 was red, E22 and E23 a little bit less.
For each plate two colonies were piked, if it was possible.
Two colonies for E9, E10, E11, E12, E17, E18, E19, E20, E21, E22, E23 plates were picked.
500 ml of LB with Ampicillin were prepared.
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July, 14th
A glycerol stock for E21-2, E23-2. E11-1 was prepared.
Cultures were saturated; plasmid purification was carried out:
| Plasmid |
DNA (ng/μl) |
| E9-1 |
114 |
| E10-1 |
72 |
| E11-1 |
78.5 |
| E12-1 |
1151.7 |
| E13-1 |
133 |
| E16-1 |
134.4 |
| E17-1 |
27.8 |
| E18-1 |
25.5 |
"
