Difference between revisions of "Team:Oxford/Notebook/Week1"
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<h4>Polymerase Chain Reaction Set-up<a name="2206c"></a></h4> | <h4>Polymerase Chain Reaction Set-up<a name="2206c"></a></h4> | ||
<br> | <br> | ||
− | <p class="text">The protocol for running a PCR using NEB’s Q5 High-Fidelity 2X Master Mix can be found <a href="http://www.neb.com/protocols/2012/08/29/protocol-for-q5-high-fidelity-2x-master-mix-m0492">here</a>. | + | <p class="text">The protocol for running a PCR using NEB’s Q5 High-Fidelity 2X Master Mix can be found <a href="http://www.neb.com/protocols/2012/08/29/protocol-for-q5-high-fidelity-2x-master-mix-m0492">here</a>.<br> |
− | + | <b><i>25µl reactions</i></b> were run, with the component breakdown by volume being:</p> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<br> | <br> | ||
<table> | <table> | ||
Line 185: | Line 181: | ||
<th>Component</th> | <th>Component</th> | ||
<th>Volume/µl</th> | <th>Volume/µl</th> | ||
+ | <th>Final concentration/nM</th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td></td> | + | <td>NEB Q5 HF Master Mix</td> |
− | <td></td> | + | <td>12.5</td> |
− | <td></td> | + | <td>-</td> |
− | <td></td> | + | </tr> |
+ | <tr> | ||
+ | <td>10µM Forward Primer</td> | ||
+ | <td>1.25</td> | ||
+ | <td>500</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10µM Reverse Primer</td> | ||
+ | <td>1.25</td> | ||
+ | <td>500</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1ng/µl<sup>-1</sup> gBlock</td> | ||
+ | <td>1.0</td> | ||
+ | <td>-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Milli-Q Water</td> | ||
+ | <td>9.0</td> | ||
+ | <td>-</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | <br> | ||
+ | |||
+ | <p class="text">* The final concentrations of the primers were noted as they are needed to determine the annealing temperatures for the primers, which can be done using NEB’s online tool at http://tmcalculator.neb.com/#!/.<br> | ||
+ | |||
+ | ** Add components in order of decreasing volume for maximum ease-of-pipetting.<br> | ||
+ | |||
+ | *** When reaction mixture is being made up, all components as well as the mixture itself are to be kept on ice, as the Master Mix is a high-fidelity polymerase that will recognize the primers as being incorrectly base-paired and be able to hydrolyse the primers if kept at room temperature.<br> | ||
+ | |||
+ | **** Use Q5 enzyme in the cold room to avoid defrosting and freezing the original stock of Q5 enzyme. This could decrease the activity of Q5 enzyme. Bring ice bucket to the cold room to bring Q5 into the bench. <br> | ||
+ | |||
+ | ***** Make sure that the primer and small amounts of DNA and primer doesn’t stick onto the side of the tube or the tip. </p> | ||
+ | |||
+ | <br> | ||
+ | <p class="text">The reaction mixture tubes were positioned in an Eppendorf Mastercycler nexus X2 and the following PCR program was run:</p> | ||
+ | <br> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Cycle(s)</th> | ||
+ | <th>Step</th> | ||
+ | <th>Temp / ℃</th> | ||
+ | <th>Time / s</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>Initial template DNA melting</td> | ||
+ | <td>98</td> | ||
+ | <td>30</td> | ||
+ | <tr> | ||
+ | <td rowspan="3">30</td> | ||
+ | <td>DNA Meting</td> | ||
+ | <td>98</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Primer Annealing</td> | ||
+ | <td>55</td> | ||
+ | <td>20</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Sequence Extension</td> | ||
+ | <td>72</td> | ||
+ | <td>60</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>Final Extension</td> | ||
+ | <td>72</td> | ||
+ | <td>120</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>Hold</td> | ||
+ | <td>4-10</td> | ||
+ | <td>-</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | <p class="text">* DNA denaturation can be performed at 98℃ because of the high thermal stability of the Q5 polymerase<br> | ||
+ | |||
+ | ** A PCR takes 20-30 seconds to extend a sequence by 1kb, and since our longest sequence is ~2kb the extension time was determined to be 60s per cycle</p> | ||
+ | <br> | ||
+ | <hr> | ||
+ | |||
<hr> | <hr> | ||
</td> | </td> |
Revision as of 13:13, 2 July 2015
22/06/2015
23/06/2015
24/06/2015
25/06/2015
22/06/2015
Date | Researcher(s) | Content | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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22/06/2015 | Whole Team |
Preparation of Stock Solutions1. gBlocksThe 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:
2. PrimersThe 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:
Preparation of Reaction Solutions1. gBlocks2µl of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20µl to make 1ng/µl-1 reaction solutions. 2. Primers2µl of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20µl to make 10µM reaction solutions. (These solutions are labelled as “Prefix primer” and “suffix primer” in eppendorf tubes in the fridge) Polymerase Chain Reaction Set-upThe protocol for running a PCR using NEB’s Q5 High-Fidelity 2X Master Mix can be found here.
* The final concentrations of the primers were noted as they are needed to determine the annealing temperatures for the primers, which can be done using NEB’s online tool at http://tmcalculator.neb.com/#!/. The reaction mixture tubes were positioned in an Eppendorf Mastercycler nexus X2 and the following PCR program was run:
* DNA denaturation can be performed at 98℃ because of the high thermal stability of the Q5 polymerase |