diff --git a/wiki/pages/notebook.html b/wiki/pages/notebook.html
index 6d0ad160b93a1197f803e0c71f19ec2a9ce3b957..c1d61270c98e136266b24ae57b49c8f0eddaeaee 100644
--- a/wiki/pages/notebook.html
+++ b/wiki/pages/notebook.html
@@ -2152,7 +2152,8 @@ A reason could be that the end Oligos didn't bind because there was not addet ad
<li>the tubes where incubate at 16 °C overnight</li>
</ul>
→ this resulted in a total of 12 tubes
-<b>The next step:</br><br>
+<br>
+<b>The next steps:</b><br>
add end oligos and run a gel to investiagte the product
</p>
</div>
@@ -2173,8 +2174,9 @@ add end oligos and run a gel to investiagte the product
<li>investigate product on a gel</li>
</ul>
<b>Results: </b><br>
-The results can be seen in the gel image. We got some bands close to 3 kb. Also, using 2 µg of the middle olgios and using a longer cooling down time in the annealing step resulted in larger fragments on average.
-<b>Next steps:</b><br>
+The results can be seen in the gel image. We got some bands close to 3 kb. Also, using 2 µg of the middle olgios and using a longer cooling down time in the annealing step resulted in larger fragments on average.<br>
+<b>Next steps:</b>
+<br>
In the next steps, 10 fragments from the gel will be isoalted in the range of 1.5 to 3 kb and the oligos extracted from the gel.
<b>Gel:</b>
@@ -2711,23 +2713,59 @@ the T4 ligase has not properly ligated start and end oligos to mid fragments, al
<h5>07.09.2024</h5>
<h3>PCR with the ligation from 05.09.</h3>
<p>
- <ul>
- <li>
- All 6 tubes and a positive control prepared
- </li>
- <li>
- Mastermix according to protocol 5.1 with 8.75 μl FW_RE_Oligo_amp-Primer and RV_RE_Oligo_amp-Primer each and a total volume of 105 μl. In each PCR tube, 15 μl and 10 μl Template were pipetted.
- </li>
- <li>
- A positive control was made as follows:
- </li>
- <li>
- - Q5 High-Fidelity 2X Master Mix: 12,5 μl
-- FW_EGFP_SLiCE_PCR: 1,25 μl
-- RV_EGFP_SLiCE_PCR: 1,25 μl
-- EGFP-Linker: 10 μl
- </li>
- </ul>
+ All 6 tubes and a positive control prepared. <br>
+ Mastermix according to protocol 5.1 with 8.75 μl FW_RE_Oligo_amp-Primer and RV_RE_Oligo_amp-Primer each and a total volume of 105 μl. In each PCR tube, 15 μl and 10 μl Template were pipetted.<br>
+ A positive control was made as follows:
+ <ul>
+ <li>Q5 High-Fidelity 2X Master Mix: 12,5 μl</li>
+ <li>FW_EGFP_SLiCE_PCR: 1,25 μl</li>
+ <li>RV_EGFP_SLiCE_PCR: 1,25 μl</li>
+ <li>EGFP-Linker: 10 μl</li>
+ </ul>
+ The annealing temperature was 56 °C and the elongation time was 3 min for 30 cycles.<br>
+ we numbered the PCR tubes from 1-6 and a +
+ <table border="1" style="width:100%; border-collapse: collapse;">
+ <tr>
+ <th style="padding: 10px; text-align: left;">Number</th>
+ <th style="padding: 10px; text-align: left;">Description</th>
+ </tr>
+ <tr>
+ <td style="padding: 10px;">1</td>
+ <td style="padding: 10px;">1,5 kb Religation from 05.09 for 48h</td>
+ </tr>
+ <tr>
+ <td style="padding: 10px;">2</td>
+ <td style="padding: 10px;">1,5 kb Religation from 05.09 for 48h</td>
+ </tr>
+ <tr>
+ <td style="padding: 10px;">3</td>
+ <td style="padding: 10px;">2 kb Religation from 05.09 for 48h</td>
+ </tr>
+ <tr>
+ <td style="padding: 10px;">4</td>
+ <td style="padding: 10px;">2 kb Religation from 05.09 for 48h</td>
+ </tr>
+ <tr>
+ <td style="padding: 10px;">5</td>
+ <td style="padding: 10px;">3 kb Religation from 05.09 for 48h</td>
+ </tr>
+ <tr>
+ <td style="padding: 10px;">6</td>
+ <td style="padding: 10px;">3 kb Religation from 05.09 for 48h</td>
+ </tr>
+ <tr>
+ <td style="padding: 10px;">+</td>
+ <td style="padding: 10px;">Positive control with EGFP+Linker and the respective primers</td>
+ </tr>
+</table>
+The PCR was checked on a 1 % agarose gel (protocol 8).
+<div style="text-align: center;">
+ <img src="https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-7-9.webp" alt="" style="width:50%;">
+ <figcaption style="font-size: 80%; color:gray;">
+ <b>Gel electrophoresis of PCR religation of oligos. 1 kb Plus DNA Ladder, 1 % agarose gel.</b>
+ </figcaption>
+</div>
+
</p>
</div>
<!--
@@ -2838,7 +2876,8 @@ the T4 ligase has not properly ligated start and end oligos to mid fragments, al
<ul>
<li>Heat the mixtures to 90°C for 5 minutes.</li>
<li>Gradually cool the mixture to room temperature over 12 minutes in the PCR block</li>
- </ul>
+ </ul><br>
+
3. Concentrations:<br>
An-Oligos: 139 µg/ 100 μl = 1.39 µg/ μl<br>
An-NdeI: 80 µg/92.16 μl = 0.868µg/ μl<br>
@@ -3180,11 +3219,6 @@ with the additional ethanol wash step before the elution. The evaporation period
Weight of the gel pieces and amount of QB buffer.
<table border="1" style="width:100%; border-collapse: collapse;">
- <tr>
- <th style="padding: 10px; text-align: left;">Produkt</th>
- <th style="padding: 10px; text-align: left;">Menge</th>
- <th style="padding: 10px; text-align: left;">Volumen</th>
- </tr>
<tr>
<td style="padding: 10px;">Resilin 2 3 kb</td>
<td style="padding: 10px;">320 mg</td>
@@ -3202,20 +3236,13 @@ Weight of the gel pieces and amount of QB buffer.
</tr>
</table>
+
Nanodrop measurement:
<table border="1" style="width:100%; border-collapse: collapse;">
- <tr>
- <th style="padding: 10px; text-align: left;">Produkt</th>
- <th style="padding: 10px; text-align: left;">Verdünnung</th>
- <th style="padding: 10px; text-align: left;">DNA + Wasser</th>
- <th style="padding: 10px; text-align: left;">Konzentration (ng/µL)</th>
- <th style="padding: 10px; text-align: left;">260/280</th>
- <th style="padding: 10px; text-align: left;">260/230</th>
- </tr>
<tr>
<td style="padding: 10px;">Resilin 2 3 kb</td>
<td style="padding: 10px;">Dilution to 1 ng/µL</td>
- <td style="padding: 10px;">4.55 µL DNA + 15.45 µL Wasser</td>
+ <td style="padding: 10px;">4.55 µL DNA + 15.45 µL water</td>
<td style="padding: 10px;">4.4 ng/µL</td>
<td style="padding: 10px;">2.26</td>
<td style="padding: 10px;">0.24</td>
@@ -3223,7 +3250,7 @@ Nanodrop measurement:
<tr>
<td style="padding: 10px;">Resilin 2 1 kb</td>
<td style="padding: 10px;">Dilution to 1 ng/µL</td>
- <td style="padding: 10px;">11.1 µL DNA + 18.9 µL Wasser</td>
+ <td style="padding: 10px;">11.1 µL DNA + 18.9 µL water</td>
<td style="padding: 10px;">18.2 ng/µL</td>
<td style="padding: 10px;">1.98</td>
<td style="padding: 10px;">0.47</td>
@@ -3231,13 +3258,14 @@ Nanodrop measurement:
<tr>
<td style="padding: 10px;">Resilin 5 1 kb</td>
<td style="padding: 10px;">Dilution to 1 ng/µL</td>
- <td style="padding: 10px;">1.7 µL DNA + 18.3 µL Wasser</td>
+ <td style="padding: 10px;">1.7 µL DNA + 18.3 µL water</td>
<td style="padding: 10px;">11.8 ng/µL</td>
<td style="padding: 10px;">1.81</td>
<td style="padding: 10px;">0.58</td>
</tr>
</table>
+
<b>PCR of the purified Resilin Oligos</b>
PCR according to protocol 5.2 with the thermofischer Phusion. Each Resilin sample should get 3 reactions with different amounts of template. (2 ng, 1 ng, 0.5 ng). Therefore 3 x 150 μl were created with 7.5 μl of the Oligo_Amp_fw and Oligo_Amp_rev primers, 3 μl dNTPs, 15 μl Phusion Buffer and 1.5 μl Phusion Polymerase. For each amount of template a different water amount was added:<br>
Mastermix 2 (2 ng): 113.5 μl water<br>
@@ -3493,7 +3521,6 @@ The purified resilin samples from the 27.09.24 were pooled (From now on listed a
First, the restriction of pET28-c and resilin was carried out with SacI and NdlI for 1 h at 37 °C. Next the samples were heat inactivated for 20 min at 65 °C.
<table border="1" style="width:100%; border-collapse: collapse;">
<tr>
- <th style="padding: 10px; text-align: left;"></th>
<th style="padding: 10px; text-align: left;">pET28-c</th>
<th style="padding: 10px; text-align: left;">Clone 1.1</th>
<th style="padding: 10px; text-align: left;">Clone 1.2</th>
@@ -3542,16 +3569,18 @@ First, the restriction of pET28-c and resilin was carried out with SacI and NdlI
<td style="padding: 10px;">-</td>
</tr>
<tr>
- <td style="padding: 10px;" colspan="6"><b>The samples had a total volume of 50 µL</b></td>
+ <td style="padding: 10px;" colspan="5"><b>The samples had a total volume of 50 µL</b>
+ </td>
</tr>
</table>
+
<table border="1" style="width:100%; border-collapse: collapse;">
<tr>
<th style="padding: 10px; text-align: left;"></th>
<th style="padding: 10px; text-align: left;">Resilin 1</th>
<th style="padding: 10px; text-align: left;">Resilin 2</th>
<th style="padding: 10px; text-align: left;">Resilin 3*</th>
- <th style="padding: 10px; text-align: left;">Resilin 3* Negative control</th>
+ <th style="padding: 10px; text-align: left;"> Negative control</th>
</tr>
<tr>
<td style="padding: 10px;">Template (µL)</td>
@@ -3601,8 +3630,7 @@ First, the restriction of pET28-c and resilin was carried out with SacI and NdlI
<th style="padding: 10px; text-align: left;">Ligation 1</th>
<th style="padding: 10px; text-align: left;">Ligation 2</th>
<th style="padding: 10px; text-align: left;">Ligation 3</th>
- <th style="padding: 10px; text-align: left;">Ligation 4</th>
- <th style="padding: 10px; text-align: left;">Negative control</th>
+ <th style="padding: 10px; text-align: left;">Ligation 4Negative control</th>
</tr>
<tr>
<td style="padding: 10px;">Ligase buffer (µL)</td>
@@ -3610,7 +3638,6 @@ First, the restriction of pET28-c and resilin was carried out with SacI and NdlI
<td style="padding: 10px;">4</td>
<td style="padding: 10px;">4</td>
<td style="padding: 10px;">4</td>
- <td style="padding: 10px;">-</td>
</tr>
<tr>
<td style="padding: 10px;">T4 Ligase (µL)</td>
@@ -3618,7 +3645,6 @@ First, the restriction of pET28-c and resilin was carried out with SacI and NdlI
<td style="padding: 10px;">0,4</td>
<td style="padding: 10px;">0,4</td>
<td style="padding: 10px;">0</td>
- <td style="padding: 10px;">-</td>
</tr>
<tr>
<td style="padding: 10px;">pET28 (µL)</td>
@@ -3626,7 +3652,6 @@ First, the restriction of pET28-c and resilin was carried out with SacI and NdlI
<td style="padding: 10px;">20 (400 ng)</td>
<td style="padding: 10px;">20 (400 ng)</td>
<td style="padding: 10px;">20 (400 ng)</td>
- <td style="padding: 10px;">-</td>
</tr>
<tr>
<td style="padding: 10px;">Resilin (µL)</td>
@@ -3634,7 +3659,6 @@ First, the restriction of pET28-c and resilin was carried out with SacI and NdlI
<td style="padding: 10px;">20 (384 ng)</td>
<td style="padding: 10px;">10 (90,8 ng)</td>
<td style="padding: 10px;">20 (N/A)</td>
- <td style="padding: 10px;">-</td>
</tr>
<tr>
<td style="padding: 10px;">ddH2O (µL)</td>
@@ -3642,7 +3666,6 @@ First, the restriction of pET28-c and resilin was carried out with SacI and NdlI
<td style="padding: 10px;">5,6</td>
<td style="padding: 10px;">15,6</td>
<td style="padding: 10px;">5,6</td>
- <td style="padding: 10px;">-</td>
</tr>
</table>
@@ -3651,8 +3674,8 @@ As a control the ligation 1 of was restricted with Sac1.
<table border="1" style="width:100%; border-collapse: collapse;">
<tr>
- <th style="padding: 10px; text-align: left;">Ligation 1</th>
<th style="padding: 10px; text-align: left;"></th>
+ <th style="padding: 10px; text-align: left;">Ligation 1</th>
</tr>
<tr>
<td style="padding: 10px;">Template (µL)</td>
@@ -3687,7 +3710,7 @@ Transformation of the recombination samples into DH5α and TOP10 using LB-Kan pl
<table border="1" style="width:100%; border-collapse: collapse;">
<tr>
- <th style="padding: 10px; text-align: left;">Ligation reaction</th>
+ <th style="padding: 10px; text-align: left;"></th>
<th style="padding: 10px; text-align: left;">DH5α</th>
<th style="padding: 10px; text-align: left;">TOP10</th>
</tr>
@@ -3729,8 +3752,8 @@ Due to too high concentrations of the pET28-c_resilin plasmid no bacteria grew o
<table border="1" style="width:100%; border-collapse: collapse;">
<tr>
- <th style="padding: 10px; text-align: left;">Ligation</th>
<th style="padding: 10px; text-align: left;"></th>
+ <th style="padding: 10px; text-align: left;">Ligation</th>
</tr>
<tr>
<td style="padding: 10px;">Ligase buffer (µL)</td>
@@ -3787,6 +3810,7 @@ Both ligation pools from the 29.09 and 30.09 were sent for a whole plasmid seque
<table border="1" style="width:100%; border-collapse: collapse;">
<tr>
<th style="padding: 10px; text-align: left;">Ligation reaction</th>
+ <th style="padding: 10px; text-align: left;"></th>
<th style="padding: 10px; text-align: left;">ddH2O</th>
<th style="padding: 10px; text-align: left;">DH5α</th>
</tr>