July, 4th
Digestions of previously purified plasmids were performed for ligations:
| Plasmid |
Kind |
Purified Dna (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume(μl) |
| <partinfo>BBa_C0060</partinfo> |
Insert |
16.5 |
4 |
1 EcoRI |
1 SpeI |
2.5 |
25 |
| <partinfo>BBa_C0061</partinfo> |
Insert |
13.2 |
7.3 |
1 XbaI |
1 PstI |
2.5 |
25 |
| <partinfo>BBa_K081022</partinfo> |
Insert |
15.7 |
4.8 |
1 EcoRI |
1 PstI |
2.5 |
25 |
| <partinfo>BBa_B0030</partinfo> |
Vector |
13.2 |
7.3 |
1 SpeI |
1 PstI |
2.5 |
25 |
| <partinfo>BBa_B0031</partinfo> |
Vector |
12.4 |
8.1 |
1 SpeI |
1 PstI |
2.5 |
25 |
| <partinfo>BBa_B0032</partinfo> |
Vector |
9.5 |
11 |
1 SpeI |
1 PstI |
2.5 |
25 |
| <partinfo>BBa_B0015</partinfo> |
Vector |
7.9 |
12.6 |
1 EcoRI |
1 XbaI |
2.5 |
25 |
| <partinfo>pSB4C5</partinfo> |
Vector |
3 |
17.5 |
1 EcoRI |
1 PstI |
2.5 |
25 |
| <partinfo>BBa_I13501</partinfo> |
Insert |
7.2 |
13.3 |
1 XbaI |
1 PstI |
2.5 |
25 |
Reaction samples were incubated at 37°C for three hours while a small-size and a medium-size agarose gel were prepared according to protocols.
In the afternoon gel electrophoresis was performed.
| [Image:UNIPV_04_07_SSgel.png] |
|
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.
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