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Exeter/28 August 2013
From 2013.igem.org
NanoDrop
We collected NanoDrop data of previously miniprepped, defrosted DNA. The NanoDrop machine was zero-ed using the elution buffer from the gel extraction kit used.
- ompC A - 20.1 ng/µl
- ompC B - 9.3ng/µl
- [http://parts.igem.org/Part:BBa_B0034?title=Part:BBa_B0034 B0034] - 26.7ng/µl
- RFP - 198ng/µl
The concentration for ompC B was too low, so we continued on to the digestion with only ompC A. The concentration for RFP was too high, so we diluted it.
- RFP (dilution) - 50.4ng/µl
Digestion
| Part | ompC (Part A) | B0034 (Part B) | AMP Plasmid | RFP (Control) |
|---|---|---|---|---|
| DNA | 12.4 ul | 9.4 ul | 10.0 ul | 5.0 ul |
| Non-nuclease Water | 3.6 ul | 6.6 ul | 5.5 ul | 11.0 ul |
| NEB Buffer | 2.5 ul | 2.5 ul | 2.5 ul | 2.5 ul |
| BSA | 0.5 ul | 0.5 ul | 0.5 ul | 0.5 ul |
| EcoRI | 0.5 ul | - | 0.5 ul | 0.5 ul |
| SpeI | 0.5 | - | - | - |
| XbaI | - | 0.5 ul | - | - |
| PstI | - | 0.5 ul | 0.5 ul | 0.5 ul |
| DpnI | - | - | 0.5 ul | - |
We pipetted the following volumes into four 0.6 ml tubes, keeping all enzymes and buffers on ice. We flicked and centrifuged the tubes to ensure mixtures we homogeneous and collected at the bottom.
The tubes were incubated at 25oC for 30 minutes and then 80oC for 20 minutes.
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 |
| ompC (R0084) | 108 |
| RBS (B0034) | 12 |
| RFP | 774 |
The ompC, B0034, AMP plasmid, and RFP were from the earlier digestions. We pippetted the following volumes into four 0.6ml tubes:
| New Part | A (ompC + B0034, eq/vol) | B (RFP control, eq/vol) | C (ompC + B0034, equimolar) | C(RFP Control, equimolar) | |
|---|---|---|---|---|---|
| Plasmid | 2 ul | 2 ul | 2 ul | 2 ul | |
| ompC | 2 ul | - | - | - | |
| ompC (10x dilution) | - | - | 1.0 ul | - | |
| B0034 | 2 ul | - | - | - | |
| B0034 (100x dilution) | - | - | 0.8 ul | - | |
| RFP | - | 2 ul | - | - | |
| RFP (10x diltion) | - | - | - | 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 | 4.7 ul | 5.8 ul |
We flicked and centrifuged the tubes to ensure mixtures we homogeneous and collected at the bottom.
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.
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