Difference between revisions of "Team:Oxford/Notebook/Week1"
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+ | |||
+ | <tr> | ||
+ | <td>23/06/2015</td> | ||
+ | <td>George, Silas,<br>Mabel, June</td> | ||
+ | <td> | ||
+ | <h4>Gel Electrophoresis of PCR-Amplified gBlocks (continued from 22/06)</h4> | ||
+ | <br> | ||
+ | <h5>For reference - gBlock sizes:</h5> | ||
+ | <br> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Label</th> | ||
+ | <th>Construct</th> | ||
+ | <th>Size / bp</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>A</td> | ||
+ | <td>LasR Holin</td> | ||
+ | <td>1763</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>B</td> | ||
+ | <td>LasR sfGFP</td> | ||
+ | <td>1829</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>C</td> | ||
+ | <td>Lsr sfGFP</td> | ||
+ | <td>910</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>D</td> | ||
+ | <td>Lsr Holin</td> | ||
+ | <td>862</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>E</td> | ||
+ | <td>DNase DsbA</td> | ||
+ | <td>646</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>F</td> | ||
+ | <td>DspB YebF</td> | ||
+ | <td>1525</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>G</td> | ||
+ | <td>DspB</td> | ||
+ | <td>1174</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>H</td> | ||
+ | <td>MccS</td> | ||
+ | <td>448</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>I</td> | ||
+ | <td>DspB Fla</td> | ||
+ | <td>1336</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>J</td> | ||
+ | <td>DspB DsbA</td> | ||
+ | <td>1228</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>K</td> | ||
+ | <td>Art-175 DsbA</td> | ||
+ | <td>1012</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>L</td> | ||
+ | <td>Art-175 YebF</td> | ||
+ | <td>1309</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>M</td> | ||
+ | <td>Art-E</td> | ||
+ | <td>644</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>N</td> | ||
+ | <td>Art-175 Fla</td> | ||
+ | <td>1120</td> | ||
+ | </tr> | ||
+ | </table> | ||
</table> | </table> | ||
Revision as of 12:51, 6 July 2015
Week 1
22/06/2015
Preparation of Stock Solutions
Preparation of Reaction Solutions
Setting up Agarose Gel for Electrophoresis of 22/06 PCR Products
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. 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 Setting up Agarose Gel for Electrophoresis for 22/06 PCR ProductsGeneral guidelines for agarose preparation:
Agarose preparation protocol: 1. Heat 2g agarose in 200ml 0.5x TBE for 2 minutes under full power in the microwave (use a 500mL Duran bottle, and place a weighing boat underneath it to prevent the causing of a mess in the event the mixture boils over; DO NOT fully tighten the Duran cap). 2. Check if the agarose has been fully dissolved. Heat it further if gel strands are visible. 3. Leave the agarose solution to cool at 50℃ for 20 minutes. 4. Pour agarose onto gel plate in a setting tray with appropriately-sized combs already fixed onto it, and leave for 20 minutes to let it set. 5. When the agarose has set, remove the combs and transfer the gel plate from the setting tray to the electrophoresis chamber. 6. Flood the gel plate with 0.5x TBE buffer up until right above the top of the wells. 7. The gel should be positioned such that the positive (red) electrode is on the far side of the gel from the wells, as the negatively-charged DNA will migrate towards the positive electrode. DNA preparation: |
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23/06/2015 | George, Silas, Mabel, June |
Gel Electrophoresis of PCR-Amplified gBlocks (continued from 22/06)For reference - gBlock sizes:
|