diff --git a/wiki/pages/notebook.html b/wiki/pages/notebook.html index 2af892097b402c7cb2f9e7ee0661cb5a53fac719..e233e3a66e94e2dc8f9b79700680e8e6142960c0 100644 --- a/wiki/pages/notebook.html +++ b/wiki/pages/notebook.html @@ -996,9 +996,11 @@ <li style="cursor: pointer;" onclick="showResilinDay('juli18')"> 18.07.2024 </li> + --> <li style="cursor: pointer;" onclick="showResilinDay('juli19')"> 19.07.2024 </li> + <!-- <li style="cursor: pointer;" onclick="showResilinDay('juli20')"> 20.07.2024 </li> @@ -1023,15 +1025,20 @@ <li style="cursor: pointer;" onclick="showResilinDay('juli27')"> 27.07.2024 </li> + --> <li style="cursor: pointer;" onclick="showResilinDay('juli28')"> 28.07.2024 </li> + <!-- <li style="cursor: pointer;" onclick="showResilinDay('juli29')"> 29.07.2024 </li> <li style="cursor: pointer;" onclick="showResilinDay('juli30')"> 30.07.2024 </li>--> + <li style="cursor: pointer;" onclick="showResilinDay('juli31')"> + 31.07.2024 + </li </ul> </div> @@ -1068,54 +1075,121 @@ --> <div class="col-lg-9 col-sm-7 col-md-8 content resilin" style="padding: 10px; display: none;" id="juli4"> <h5>04.07.2024</h5> - <h3>Repeating the Quick Change mutagenesis</h3> + <h3>Doing a gradient PCR for Quick Change mutagenesis</h3> <p> + Repeating the Quick Change mutagenesis using a longer elongation time and a temperature gradient to find the perfect annealing temperature. + <br> + template: + <table border="1" cellpadding="5" style="border-collapse: collapse; width: 100%;"> + <tbody> + <tr style="font-weight: bold; text-align: center;"> + <td></td> + <td>BL21</td> + <td>Top10</td> + </tr> + <tr style="text-align: center;"> + <td style="font-weight: bold;">OD</td> + <td>0,03</td> + <td>0,033</td> + </tr> + </tbody> + </table> + Thermocycling Conditions: + <table border="1" cellpadding="5" style="border-collapse: collapse; width: 100%;"> + <tbody> + <tr style="font-weight: bold; text-align: center;"> + <td>STEP</td> + <td>Temp</td> + <td>Time</td> + <td>Cyles</td> + </tr> + <tr style="text-align: center;"> + <td style="font-weight: bold;">annealing (gradient)</td> + <td>50°C</td> + <td>30 seconds</td> + <td>30</td> + </tr> + <tr style="text-align: center;"> + <td style="font-weight: bold;">elongation</td> + <td>72°C</td> + <td>120 seconds</td> + <td>30</td> + </tr> + </tbody> + </table> + For annealing temperature: + <table border="1" cellpadding="5" style="border-collapse: collapse; width: 100%;"> + <tbody> + <tr style="font-weight: bold; text-align: center;"> + <td>lane</td> + <td>1</td> + <td>2</td> + <td>3</td> + <td>4</td> + <td>5</td> + <td>6</td> + <td>7</td> + <td>8</td> + <td>9</td> + <td>10</td> + </tr> + <tr style="text-align: center;"> + <td style="font-weight: bold;">sample</td> + <td>DNA Ladder</td> + <td>1</td> + <td>2</td> + <td>3</td> + <td>4</td> + <td>5</td> + <td>6</td> + <td>7</td> + <td>8</td> + <td>DNA Ladder</td> + </tr> + <tr style="text-align: center;"> + <td style="font-weight: bold;">annealing temp.</td> + <td>-</td> + <td>50 °C</td> + <td>53 °C</td> + <td>56 °C</td> + <td>59 °C</td> + <td>63 °C</td> + <td>66 °C</td> + <td>69 °C</td> + <td>72 °C</td> + <td>-</td> + </tr> + </tbody> + </table> + Elongation time was 120 seconds + <br> + <b>Results:</b><br> + The lids popped of, so there was only one PCR to be seen in the gel (lane3). + <br> + <div style="text-align: center;"> + <img src="https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-4-7.webp" alt="" style="width:50%;"> + <figcaption style="font-size: 80%; color:gray;"> + <b>Site directed mutagenesis of pMK1_RE4.</b> + A new PCR with a gradient of different Annealing temperatures (see table above) and a longer elongation time. The Site directed mutagenesis was performed on the plasmid pMK1_RE4-1:3_4; Ladder: 1 kb Plus; Agarose gel (1 %). + </figcaption> + </div> + <b>Problem:</b> + The lids were not closed properly + <br> + + <b>Solution:</b><br> <ul> <li> - A Quick change mutagenesis of pMK1 was performed using a longer elongation time and a temperature gradient to find the perfect annealing temperature. + we ordered new primers which should bind better </li> - </ul> - Primer: - <ul> <li> - FW_pMK1_RE4_G->C + we also ordered primes to remove the second BsmBI-Binding sequence behind the poly-A tale </li> <li> - RW_pMK1_RE4_G->C + we would also try a new method to generate repeats called the rolling circle method where circular DNA containing only 4 repeats of resilin is generated. Than a phi29 polymerase will circulate around this circular DNA generating more repeats. </li> </ul> - Template: - <ul> - <li> - pMK1_RE4_1:3_4 - </li> - </ul> - Thermocycling conditions: - <table> - <thead> - <tr> - <th>Step</th> - <th>Temperature [°C]</th> - <th>Time [s]</th> - <th>Cycles</th> - </tr> - </thead> - <tbody> - <tr> - <td>annealing</td> - <td>50</td> - <td>30</td> - <td>30</td> - </tr> - <tr> - <td>elongation</td> - <td>72</td> - <td>>120</td> - <td>30</td> - </tr> - </tbody> - </table> - The lids popped of so the contents evaporated. + </p> </div> <!--