Exeter/30 August 2013
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== Results from yesterday's transformations == | == Results from yesterday's transformations == | ||
- | + | <i>ompF</i> + RBS 100 µl equal amounts - 44 colonies | |
- | + | <i>ompF</i> + RBS 100 µl equal moles - 8 colonies | |
- | + | <i>ompF</i> + RBS 400 µl equal amounts - 107 colonies | |
- | + | <i>ompF</i> + RBS 400 µl equal moles - 35 colonies | |
- | RFP control equal amounts - 15 | + | [http://parts.igem.org/Part:BBa_J04450 RFP control] equal amounts - 15 colonies |
- | + | ||
- | + | ||
+ | [http://parts.igem.org/Part:BBa_J04450 RFP control] equal moles - 49 colonies | ||
==Ligation== | ==Ligation== | ||
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- | K592011, B0015, AMP plasmid, and RFP were from the | + | K592011, B0015, AMP plasmid, and RFP were from the digestions on the 29th. |
- | We | + | We pipetted the following volumes into four 0.6ml tubes: |
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| Plasmid || 2 ul || 2 ul || 2 ul || 2 ul | | Plasmid || 2 ul || 2 ul || 2 ul || 2 ul | ||
|- | |- | ||
- | | Cyan || 2 ul || - || | + | | Cyan || 2 ul || - || 0.66 || - |
- | + | ||
- | + | ||
|- | |- | ||
| B0015 || 2 ul || - || - || - | | B0015 || 2 ul || - || - || - | ||
|- | |- | ||
- | | B0015 ( | + | | B0015 (10x dilution) || - || - || 1.2 ul || - |
|- | |- | ||
- | | RFP || - || 2 ul | + | | RFP || - || 2 ul || - || 0.74 ul |
- | + | ||
- | + | ||
|- | |- | ||
| T4 ligase buffer || 1.0 ul || 1.0 ul || 1.0 ul || 1.0 ul | | T4 ligase buffer || 1.0 ul || 1.0 ul || 1.0 ul || 1.0 ul | ||
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| T4 DNA ligase || 0.5 ul || 0.5 ul || 0.5 ul || 0.5 ul | | T4 DNA ligase || 0.5 ul || 0.5 ul || 0.5 ul || 0.5 ul | ||
|- | |- | ||
- | | Non-nuclease water || 2.5 ul || 4.5 ul || 4. | + | | Non-nuclease water || 2.5 ul || 4.5 ul || 4..64 ul || 5.76 ul || |
+ | |} | ||
+ | |||
+ | |||
+ | The tubes were incubated at 16<sup>o</sup>C for 30 minutes and then 80<sup>o</sup>C for 20 minutes. | ||
+ | |||
+ | ==Gel of Digestion== | ||
+ | |||
+ | While carrying out the ligation, we ran a gel of our digested parts. | ||
+ | |||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | !Lane !! Content | ||
+ | |- | ||
+ | | 1 || 1kb Gene Ruler | ||
+ | |- | ||
+ | | 2 || K592011 | ||
+ | |- | ||
+ | | 3 || B0015 | ||
+ | |- | ||
+ | | 4 || AMP Plasmid | ||
+ | |- | ||
+ | | 5 || RFP | ||
+ | |- | ||
+ | | 6 || 1kb Gene Ruler | ||
|} | |} | ||
- | + | Take me back to the [https://2013.igem.org/Team:Exeter/Notebook notebook]. | |
- | + | </div> | |
+ | </div> | ||
+ | {{:Team:Exeter/Template/Footer}} |
Latest revision as of 20:39, 2 October 2013
Results from yesterday's transformations
ompF + RBS 100 µl equal amounts - 44 colonies
ompF + RBS 100 µl equal moles - 8 colonies
ompF + RBS 400 µl equal amounts - 107 colonies
ompF + RBS 400 µl equal moles - 35 colonies
[http://parts.igem.org/Part:BBa_J04450 RFP control] equal amounts - 15 colonies
[http://parts.igem.org/Part:BBa_J04450 RFP control] equal moles - 49 colonies
Ligation
The ligation was carried out using two methods: equal volumes and equal moles. To calculate the equimolar volumes, we used the following data:
Part | Part Length (bp) |
---|---|
AMP plasmid | 2155 |
K592011 | 702 |
RBS (B0015) | 129 |
RFP | 774 |
K592011, B0015, AMP plasmid, and RFP were from the digestions on the 29th. We pipetted the following volumes into four 0.6ml tubes:
New Part | A (Cyan + B0015, eq/vol) | B (RFP control, eq/vol) | C (Cyan + B0015, equimolar) | C(RFP Control, equimolar) | |
---|---|---|---|---|---|
Plasmid | 2 ul | 2 ul | 2 ul | 2 ul | |
Cyan | 2 ul | - | 0.66 | - | |
B0015 | 2 ul | - | - | - | |
B0015 (10x dilution) | - | - | 1.2 ul | - | |
RFP | - | 2 ul | - | 0.74 ul | |
T4 ligase buffer | 1.0 ul | 1.0 ul | 1.0 ul | 1.0 ul | |
T4 DNA ligase | 0.5 ul | 0.5 ul | 0.5 ul | 0.5 ul | |
Non-nuclease water | 2.5 ul | 4.5 ul | 4..64 ul | 5.76 ul |
The tubes were incubated at 16oC for 30 minutes and then 80oC for 20 minutes.
Gel of Digestion
While carrying out the ligation, we ran a gel of our digested parts.
Lane | Content |
---|---|
1 | 1kb Gene Ruler |
2 | K592011 |
3 | B0015 |
4 | AMP Plasmid |
5 | RFP |
6 | 1kb Gene Ruler |
Take me back to the notebook.