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Exeter/28 August 2013
From 2013.igem.org
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| - | ==NanoDrop== | + | {{:Team:Exeter/Template/Header}} |
| + | <div class="container"> | ||
| + | <div class="row"> | ||
| + | <div class="span" style="text-align:justify"> | ||
| + | |||
| + | ==NanoDrop== __NOTOC__ | ||
We collected NanoDrop data of previously miniprepped, defrosted DNA. | 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. | The NanoDrop machine was zero-ed using the elution buffer from the gel extraction kit used. | ||
| - | * | + | *<i>ompC</i> A - 20.1 ng/µl |
| - | * | + | *<i>ompC</i> B - 9.3ng/µl |
| - | *B0034 - 26.7ng/ | + | *[http://parts.igem.org/Part:BBa_B0034?title=Part:BBa_B0034 B0034] - 26.7ng/µl |
| - | *RFP - 198ng/ | + | *RFP - 198ng/µl |
| - | The concentration for | + | The concentration for <i>ompC</i> B was too low, so we continued on to the digestion with only <i>ompC</i> A. |
The concentration for RFP was too high, so we diluted it. | The concentration for RFP was too high, so we diluted it. | ||
| - | *RFP (dilution) - 50.4ng/ | + | *RFP (dilution) - 50.4ng/µl |
==Digestion== | ==Digestion== | ||
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{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
| - | ! Part !! | + | ! Part !! <i>ompC</i> (Part A) !! B0034 (Part B) !! AMP Plasmid !! RFP (Control) |
|- | |- | ||
| DNA || 12.4 ul || 9.4 ul || 10.0 ul || 5.0 ul | | DNA || 12.4 ul || 9.4 ul || 10.0 ul || 5.0 ul | ||
| Line 29: | Line 34: | ||
| BSA || 0.5 ul || 0.5 ul || 0.5 ul || 0.5 ul | | BSA || 0.5 ul || 0.5 ul || 0.5 ul || 0.5 ul | ||
|- | |- | ||
| - | | | + | | <i>EcoRI</i> || 0.5 ul || - || 0.5 ul || 0.5 ul |
|- | |- | ||
| - | | | + | | <i>SpeI</i> || 0.5 || - || - || - |
|- | |- | ||
| - | | | + | | <i>XbaI</i> || - || 0.5 ul || - || - |
|- | |- | ||
| - | | | + | | <i>PstI</i> || - || 0.5 ul || 0.5 ul || 0.5 ul |
|- | |- | ||
| - | | | + | | <i>DpnI</i> || - || - || 0.5 ul || - |
|} | |} | ||
| Line 43: | Line 48: | ||
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. | 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 | + | The tubes were incubated at 25<sup>o</sup>C for 30 minutes and then 80<sup>o</sup>C for 20 minutes. |
==Ligation== | ==Ligation== | ||
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| AMP plasmid || 2155 | | AMP plasmid || 2155 | ||
|- | |- | ||
| - | | | + | | <i>ompC</i> (R0084) || 108 |
|- | |- | ||
| RBS (B0034) || 12 | | RBS (B0034) || 12 | ||
| Line 64: | Line 69: | ||
| - | The | + | The <i>ompC</i>, B0034, AMP plasmid, and RFP were from the earlier digestions. |
We pippetted the following volumes into four 0.6ml tubes: | We pippetted the following volumes into four 0.6ml tubes: | ||
| Line 70: | Line 75: | ||
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
| - | ! New Part !! A ( | + | ! New Part !! A (<i>ompC</i> + B0034, eq/vol) !! B (RFP control, eq/vol) !! C (<i>ompC</i> + B0034, equimolar) !! C(RFP Control, equimolar) |
|- | |- | ||
| Plasmid || 2 ul || 2 ul || 2 ul || 2 ul | | Plasmid || 2 ul || 2 ul || 2 ul || 2 ul | ||
|- | |- | ||
| - | | | + | | <i>ompC</i> || 2 ul || - || - || - |
|- | |- | ||
| - | | | + | | <i>ompC</i> (10x dilution) || - || - || 1.0 ul || - |
|- | |- | ||
| B0034 || 2 ul || - || - || - | | B0034 || 2 ul || - || - || - | ||
| Line 95: | Line 100: | ||
We flicked and centrifuged the tubes to ensure mixtures we homogeneous and collected at the bottom. | We flicked and centrifuged the tubes to ensure mixtures we homogeneous and collected at the bottom. | ||
| - | The tubes were incubated at | + | 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== | ==Gel of Digestion== | ||
While carrying out the ligation, we ran a gel of our digested parts. | While carrying out the ligation, we ran a gel of our digested parts. | ||
| + | |||
| + | Take me back to the [https://2013.igem.org/Team:Exeter/Notebook notebook]. | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | {{:Team:Exeter/Template/Footer}} | ||
Latest revision as of 20:53, 2 October 2013
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.
Take me back to the notebook.
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