Difference between revisions of "Team:Oxford/Test/Notebook"
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The gBlocks were made into 10ng/µl stock solutions in Milli-Q water for storage: | The gBlocks were made into 10ng/µl stock solutions in Milli-Q water for storage: | ||
| + | <table border="1"> | ||
<tr> | <tr> | ||
<td>mass/ng</td> | <td>mass/ng</td> | ||
<td>conc/ng\(\mu\)l<sup>-1</sup></td> | <td>conc/ng\(\mu\)l<sup>-1</sup></td> | ||
| − | <td>final volume\(mu\)l</td> | + | <td>final volume\(\mu\)l</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
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<td>20</td> | <td>20</td> | ||
</tr> | </tr> | ||
| + | </table> | ||
</p> | </p> | ||
</li> | </li> | ||
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The primers were made into 100µM stock solutions in Milli-Q water for storage: | The primers were made into 100µM stock solutions in Milli-Q water for storage: | ||
| + | <table border="1"> | ||
<tr> | <tr> | ||
<td>amt/10<sup>-9</sup>mol</td> | <td>amt/10<sup>-9</sup>mol</td> | ||
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<td>343</td> | <td>343</td> | ||
</tr> | </tr> | ||
| + | </table> | ||
</p> | </p> | ||
</li> | </li> | ||
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<h4>Polymerase Chain Reaction Set-Up</h4> | <h4>Polymerase Chain Reaction Set-Up</h4> | ||
<p> | <p> | ||
| − | The protocol for running a PCR using NEB's Q5 High-Fidelity 2X Master Mix can be found <a href="https://www.neb.com/protocols/2012/08/29/protocol-for-q5-high-fidelity-2x-master-mix-m0492">here</a> | + | The protocol for running a PCR using NEB's Q5 High-Fidelity 2X Master Mix can be found <a href="https://www.neb.com/protocols/2012/08/29/protocol-for-q5-high-fidelity-2x-master-mix-m0492">here.</a> |
</p> | </p> | ||
<p> | <p> | ||
25\(\mu\)l reactions were run, with the volume breakdown by component being: | 25\(\mu\)l reactions were run, with the volume breakdown by component being: | ||
| + | <table border="1"> | ||
<tr> | <tr> | ||
<td>Component</td> | <td>Component</td> | ||
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<td>-</td> | <td>-</td> | ||
</tr> | </tr> | ||
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</p> | </p> | ||
</div> | </div> | ||
Revision as of 13:52, 13 August 2015
Notebook
Week 1
Week 1 - Day 1
Preparation of Stock Solutions
-
gBlocks
The gBlocks ordered from IDT arrived in the form of vials of 200ng solid DNA powder.
(refer to BioBricks page for information on DNA sequences)
The gBlocks were made into 10ng/µl stock solutions in Milli-Q water for storage:
mass/ng conc/ng\(\mu\)l-1 final volume\(\mu\)l 200 10 20 -
Primers
The forward and reverse primers ordered from IDT came in 32.4nmol and 34.3nmol of solid respectively.
(Sequences: Forward - CTTTTTTGCCGGACTGC Reverse - ATGATTTCTGGAATTCGC)
The primers were made into 100µM stock solutions in Milli-Q water for storage:
amt/10-9mol conc/10-6M final volume/10-6L 32.4 100 324 34.3 100 343
Preparation of Reaction Solutions
-
gBlocks
2\(\mu\)l of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20\(\mu\)l to make 1ng/\(\mu\)l-1
-
Primers
2\(\mu\)l of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20\(\mu\)l to make 10\(\mu\)M reaction solutions.(The solutions are labelled as "Prefix primer" and "suffix primer" in eppendorf tubes in the fridge)
Polymerase Chain Reaction Set-Up
The protocol for running a PCR using NEB's Q5 High-Fidelity 2X Master Mix can be found here.
25\(\mu\)l reactions were run, with the volume breakdown by component being:
| Component | Volume/\(\mu\)l | Final conc/nM |
| Q5 HF Master Mix | 12.5 | - |
| 10\(\mu\)M Forward Primer | 1.25 | 500 |
| 10\(\mu\)M Reverse Primer | 1.25 | 500 |
| 1ng/\(\mu\)l-1 | 1.0 | - |
| Milli-Q Water | 9.0> | - |
{:Team:Oxford/Templates/Foot}}