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Notebook Table [mRNA Entries, unfinished]

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<script>
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document.addEventListener('DOMContentLoaded', () => {
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new NotebookPage("Plasmid-Amplification");
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new NotebookPage("mRNA-Purification");
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<!-- Notebook table -->
<div style="position: relative;">
<table>
<tr style="height: 75px; font-weight: bold;">
<td class="left-column">2024</td>
<th>mRNA</th>
<th>LNP's</th>
<th>Cell Line Experiments</th>
<th>Nasal Spray</th>
<th>Interactions with Local Communities</th>
</tr>
<tr>
<td class="left-column">April</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td class="left-column">May</td>
<td id="PCR-Amplification"> PCR Amplification of Factor VIII and GFP </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td class="left-column">June</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td class="left-column">July</td>
<td><table><tr>
<td class="third-cell" id="Plasmid-Amplification"> Plasmid Amplification of FVIII </td>
<td class="third-cell" id="In-vitro"> In vitro Transcription </td>
<td class="third-cell" id="mRNA-Purification"> mRNA purification </td>
</tr></table></td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td class="left-column">Aug.</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
</table>
</div>
<!-- Notebook page display -->
<div>
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<h1 id="task" style="text-align: center;">Task</h1>
<p id="datetime" style="text-align:center;">Time: xx:xx xx/xx/xx</p>
<h2 style="text-align: center;">All Participants:</h2>
<ul id="participants">
<li>Scientist 1</li>
<li>Researcher 2</li>
<li>Data collector 3</li>
</ul>
</div>
<div id="scroller" style="background-color: white; color: black; flex: 2; overflow-y:auto; padding:5px;">
<div id="journal">
<p>
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Ullamcorper dignissim cras tincidunt lobortis feugiat vivamus. Accumsan sit amet nulla facilisi morbi tempus iaculis urna. Diam maecenas sed enim ut sem viverra aliquet eget. Malesuada proin libero nunc consequat interdum. Pharetra massa massa ultricies mi quis hendrerit dolor magna. Euismod elementum nisi quis eleifend quam adipiscing vitae proin sagittis. Blandit cursus risus at ultrices. Dui sapien eget mi proin sed libero enim sed. Nisl suscipit adipiscing bibendum est ultricies integer. Sapien pellentesque habitant morbi tristique senectus et netus et malesuada. Arcu vitae elementum curabitur vitae nunc sed. Volutpat sed cras ornare arcu dui vivamus arcu felis.
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<!-- Notebook page data -->
<div>
<div id="page-example" class="retrievable">
<p id="example-task">
<!-- Insert the name of the notebook page page riight there. -->
Name of Notebook Task/Page
</p>
<p id="example-datetime">
<!-- Insert the time(s) below this comment-->
01/01/1970 - 21/12/12
</p>
<ul id="example-participants">
<!-- Insert each participant within its own <li></li> tag below.-->
<li>Participant 1</li>
<li>...</li>
<li>Participant n</li>
</ul>
<ul id="example-protocols">
<!-- Insert each protocol within its own <li></li> tag below.-->
<li>Protocol 1</li>
<li>...</li>
<li>Protocol n</li>
</ul>
<div id="example-journal">
<!-- Insert the actual lab notes here. -->
<table>
<tr><td class="journal-td">
<p>0th of Nulluary 0000</p>
<h3>1st Entry</h3>
<h4>Participants:</h4>
<ul>
<li>Participants</li>
<li>Go</li>
<li>Here!!</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li>Protocols</li>
<li>Go</li>
<li>Here!!</li>
</ul>
<h4>Experimental:</h4>
<p>
This is a journal entry, where something happens.
</p>
<p>
And that's that!
</p>
</td></tr>
<tr><td class="journal-td">
<p>0th of Nulluary 0000</p>
<h3>2nd Entry</h3>
<h4>Participants:</h4>
<ul>
<li>Participants</li>
<li>Go</li>
<li>Here!!</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li>Protocols</li>
<li>Go</li>
<li>Here!!</li>
</ul>
<h4>Experimental:</h4>
<p>
This is a journal entry, where something happens.
</p>
<p>
And that's that!
</p>
</td></tr>
</table>
</div></div>
<!-- mRNA Cells -->
<div id="page-PCR-Amplification" class="retrievable">
<p id="PCR-Amplification-task">
PCR Amplification of Factor VIII and GFP
</p>
<p id="PCR-Amplification-datetime">
<!-- Insert the time(s) below this comment-->
16/05/24 - 03/06/24
</p>
<ul id="PCR-Amplification-participants">
<!-- Insert each participant within its own <li></li> tag below.-->
<li>Henriete</li>
<li>Thijmen</li>
<li>Vasilis</li>
</ul>
<div id="PCR-Amplification-journal">
<table>
<tr><td class="journal-td">
<p>16th of May 2024</p>
<h3>PCR Amplification, 1st Attempt</h3>
<h4>Participants:</h4>
<ul>
<li>Henriete</li>
<li>Thijmen</li>
<li>Vasilis</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><a href="https://static.igem.wiki/teams/5342/documents/protocols/pcr-procedure-radboud-university-team.pdf" target="_blank">
Factor 8 Plasmid Amplification
</a></li>
</ul>
<h4>Experimental:</h4>
<p>
3 PCR reactions of 20 uL were prepared
The primers used were: T7 terminator forward primer and F8 primer reverse primer. (link to IDT page or provide sequences?)
The test reaction was made with 1 uL of F8 DNA template
The positive control was made with 1 uL of PET 28 P450 BM3 No2
The negative control was made with 1uL MilliQ.
The PCR reaction was run for 30 cycles.
</p>
</td></tr>
<tr><td class="journal-td">
<p>24th of May 2024</p>
<h3>PCR Product Analysis</h3>
<h4>Participants:</h4>
<ul>
<li>Henriete</li>
<li>Vasilis</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><a href="https://static.igem.wiki/teams/5342/documents/protocols/denaturing-agarose-gel-electrophoresis.pdf" target="_blank">
Native Agarose Gel Electrophoresis of RNA
</a></li>
</ul>
<h4>Experimental:</h4>
<p>
Gel electrophoresis was performed with 10 uL of the three PCR reactions.
The gel was run at 110V for 50 min.
</p>
<img class="journal-img" src="https://static.igem.wiki/teams/5342/images/notebook/mrna/pcr-amplification-1.webp" alt="PCR Amplification Image">
<h4>Discussion:</h4>
<p>
The positive and negative controls are as expected.
For the F8 reaction, we can see little smears and some nice bands.
The amplified F8 DNA was expected around 7000 bp, for which we do see a small band, but the band around 1000 bp is not the desired product.
Based on these results we decided to extract the DNA out of the gel and amplify it in order to get a lot of ‘pure’ factor 8 DNA.
</p>
</td></tr>
<tr><td class="journal-td">
<p>3rd of June 2024</p>
<h3>DNA Extraction from Gel</h3>
<h4>Participants:</h4>
<ul>
<li>Henriete</li>
<li>Vasilis</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><a href="https://static.igem.wiki/teams/5342/documents/protocols/qiagen-gel-extraction-protocol.pdf" target="_blank">
QIAGEN Gel Extraction Protocol
</a></li>
</ul>
<h4>Experimental:</h4>
<p>
This part is not completely clear to me…
</p>
</td></tr>
</table>
</div>
</div>
<div id="page-Plasmid-Amplification" class="retrievable">
<p id="Plasmid-Amplification-task">
Plasmid Amplification of FVIII
</p>
<p id="Plasmid-Amplification-datetime">
03/07/24 - 08/07/24
</p>
<ul id="Plasmid-Amplification-participants">
<li>Kate</li>
<li>Lea</li>
</ul>
<div id="Plasmid-Amplification-journal">
<table>
<tr><td class="journal-td">
<p>3rd of July 2024 - 4th of July 2024</p>
<h3>Plasmid Cloning</h3>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><p style="background-color:yellow">
Link to plasmid cloning protocol (?) <!-- Insert Protocol -->
</p></li>
</ul>
<h4>Experimental:</h4>
<p>
200 uL of XL1Blue and 200 uL Top10 bacteria were transfected with 1uL FVIII plasmid (link to the plasmid) following the cloning protocol.
After incubating the bacterial plates for 24 h, the Top10 plate had many miniscule colonies, while Xl1Blue had fewer, but larger colonies.
6 colonies were picked from the XL1Blue plate and 4 from the Top10 plate.
In total 10 5mL liquid cultures were prepared and incubated overnight at 37°C.
</p>
</td></tr>
<tr><td class="journal-td">
<p>5th of July 2024</p>
<h3>PCR Product Analysis</h3>
<h4>Participants:</h4>
<ul>
<li>Kate</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><p style="background-color:yellow">
link to miniprep protocol (??) <!-- Insert Protocol -->
</p></li>
<li><p style="background-color:yellow">
link to enzyme digestion protocol (??) <!-- Insert Protocol -->
</p></li>
<li><a href="https://static.igem.wiki/teams/5342/documents/protocols/denaturing-agarose-gel-electrophoresis.pdf" target="_blank">
Native Agarose Gel Electrophoresis of RNA
</a></li>
</ul>
<h4>Experimental:</h4>
<p>
A miniprep was performed on 2mL of all 10 liquid cultures.
2.5 uL of each XL1Blue plasmids and 5 uL of the Top10 plasmids were degraded with Xho1 and EcoRI and analysed via agarose gel electrophoresis.
</p>
<h4>Results and Discussion:</h4>
<p>EcoRI digestion:</p>
<img class="journal-img" src="https://static.igem.wiki/teams/5342/images/notebook/mrna/plasmid-amplification-1.webp" alt="Plasmid Amplification">
<p>
The EcoRI digestion is to check the structure of the plasmid.
Four bands were expected (347bp, 1725bp, 4688bp and 5323bp) and that is also visible on the gel.
</p>
<p>Xho1 digestion:</p>
<img class="journal-img" src="https://static.igem.wiki/teams/5342/images/notebook/mrna/plasmid-amplification-2.webp" alt="Plasmid Amplification">
<p>
Meant to linearise the plasmid. One band is visible with the length of the whole plasmid (12.1kbp) as expected.
Both digestions look good, the next step is to do a larger scale Xho1 digestion to collect more linearised plasmid for in vitro transcription.
</p>
</td></tr>
<tr><td class="journal-td">
<p>8th of July 2024</p>
<h3>Large Xho1 digestion and DNA purification</h3>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><p style="background-color:yellow">
link to enzyme digestion protocol (??)
</p></li>
<li><p style="background-color:yellow">
link to DNA purification column protocol (??)
</p></li>
</ul>
<h4>Experimental:</h4>
<p>
4 enzyme digestions with a volume of 150 uL were prepared using plasmids isolated from XL1Blue.
11.9 uL pcDNA was used to reach a final concentration of 9ng.
The digested plasmids were purified using a spin column.
The concentrations of the purified product were measured to be 79.9, 96.6, 94.7 and 88.8 ng/uL.
The purified products were put on gel (110V 50 min) to check the quality.
</p>
<p>
Note: the samples were floating out of the wells, so we prepared them again with a 5:2 sample to dye (double the amount of dye)
</p>
<h4>Results and Discussion:</h4>
<img class="journal-img" src="https://static.igem.wiki/teams/5342/images/notebook/mrna/plasmid-amplification-3.webp" alt="Plasmid Amplification">
<p>
The digested plasmids show clear bands at the length where we expected them and no large smears, so we can move on to transcription.
</p>
</td></tr>
</table>
</div>
</div>
<div id="page-In-vitro" class="retrievable">
<p id="In-vitro-task">
In vitro Transcription
</p>
<p id="In-vitro-datetime">
09/07/24 - 10/07/24
</p>
<ul id="In-vitro-participants">
<li>Lea</li>
<li>Thijmen</li>
</ul>
<div id="In-vitro-journal">
<table>
<tr><td class="journal-td">
<p>9th of July 2024</p>
<h3>First Trial IVT of eGFP and Factor VIII RNA</h3>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><a href="https://static.igem.wiki/teams/5342/documents/protocols/in-vitro-synthesis-of-rna-using-t7-rna-polymerase.pdf">
<i>in vitro</i> synthesis of RNA using T7 RNA polymerase
</a></li>
<li><a href="https://static.igem.wiki/teams/5342/documents/protocols/denaturing-agarose-gel-electrophoresis.pdf">
Native Agarose Gel Electrophoresis of RNA
</a></li>
</ul>
<h4>Experimental:</h4>
<p>
10 epps with each 20uL mastermix were prepared.
1uL from 5 different DNA templates (F8 1-4 pcDNA see F8 cloning and GFP cDNA see GFP PCR) to reach an approximate final concentration of 10nM.
For every different DNA sample, one epp got 2uL mutated (look up specific type) T7 polymerase and the other 4uL.
5uL of all samples was mixed with 2uL loading dye and run on an agarose gel (110V 40 min).
</p>
<img class="journal-img" src="https://static.igem.wiki/teams/5342/images/notebook/mrna/in-vitro-1.webp" alt="in vitro">
<p>
We try to recycle our gels as much as possible to reduce waste, the crossed out lanes are from a different experiment (refer to FVIII plasmid purification).
</p>
<h4>Discussion:</h4>
<p>
It is weird that 1, 2 and 4 with 2uL T7 did not show a lot of transcription while 3 seems to have a lot, because they should practically be the same. Overall, the reaction seems to work better with 4uL T7, so we will continue with that.
The FVIII RNA was expected to be longer (7000 instead of 1200). This could be the result of RNA degradation or self splicing, but it could also be because of the properties of the gel and because RNA is not linear.
In addition, the GFP RNA did not work out that well in both cases. it could have something to do with the template. It turned out that the calculations on the amount of GFP template were incorrect, so in the next experiment the correct amount should be added.
</p>
</td></tr>
<tr><td class="journal-td">
<p>10th of July 2024</p>
<h3>Second Trial IVT of eGFP and FVIII RNA</h3>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
<li>Thijmen</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><a href="https://static.igem.wiki/teams/5342/documents/protocols/in-vitro-synthesis-of-rna-using-t7-rna-polymerase.pdf">
<i>in vitro</i> synthesis of RNA using T7 RNA polymerase
</a></li>
<li><a href="https://static.igem.wiki/teams/5342/documents/protocols/denaturing-agarose-gel-electrophoresis.pdf">
Native Agarose Gel Electrophoresis of RNA
</a></li>
</ul>
<h4>Experimental:</h4>
<p>Overview:</p>
<table>
<tr>
<td>EFGP + mutT7 = 9</td>
<td>EFGP + mutT7 + RNAase inh. = 10</td>
<td>EGFP + WT T7 = 4</td>
<td>EGFP + WT T7 + RNAase inh. = 5</td>
</tr>
<tr>
<td></td>
<td>F8 -1 + mut T7 + RNAase inh = 3</td>
<td>F8 - 1 + WT T7 = 1</td>
<td>F8 - 1 + WT T7 + RNAase inh. = 5</td>
</tr>
<tr>
<td></td>
<td> F8-4+mut T7 + RNAase inh = 8</td>
<td>F8 - 4 + WT T7 = 6</td>
<td>F8 - 4 + WT T7 + RNAase inh. = 7</td>
</tr>
</table>
<p>
10 epps with 20 uL IVT mastermix were prepared.
If both 4uL T7 was used for both varieties.
1 uL of F8 DNA template was used from both template samples, 1.5 uL of the diluted GFP template was used to reach the right amount of template.
For samples containing RNase inhibitor, 0.5 uL ribolock was used.
</p>
<img class="journal-img" src="https://static.igem.wiki/teams/5342/images/notebook/mrna/in-vitro-2.webp" alt="in vitro">
<h4>Discussion:</h4>
<p>
First important to note is that during the pipetting of well 3 the pipet point fell, so any abnormalities could be linked to that.
When comparing well 7 and 8, the effect of RNase inhibitor is shown clearly.
Compared to the previously used mutated T7, the WT T7 shows a higher yield for both FVIII RNA and EGFP RNA.
The bands are still very smeared, especially the FVIII RNAs, either due to RNA secondary structures or low purity.
To investigate this, a denaturing agarose gel should be performed.
In addition, transcription using WT T7 and RNase inhibitor can be scaled up.
</p>
</td></tr>
</table>
</div>
</div>
<div id="page-mRNA-Purification" class="retrievable">
<p id="mRNA-Purification-task">
mRNA Purification
</p>
<p id="mRNA-Purification-datetime">
18/07/24 - 23/07/24
</p>
<ul id="mRNA-Purification-participants">
<li>Andreas</li>
<li>Lea</li>
<li>Maks</li>
<li>Thijmen</li>
</ul>
<div id="mRNA-Purification-journal">
<table>
<tr><td class="journal-td">
<p>18th of July 2024</p>
<h3>1st Entry</h3>
<h4>1st Trial RNA Purification using phenol:chloroform</h4>
<ul>
<li>Adrian</li>
<li>Lea</li>
<li>Maks</li>
<li>Thijmen</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><p style="background-color:yellow">
Protocol based on <a href="https://www.protocols.io/view/rna-clean-up-by-phenol-chloroform-yxmvmw46g3pe/v2?step=null">this</a> (??) <!-- Insert Protocol -->
</p></li>
</ul>
<h4>Experimental:</h4>
<ol>
<li>20uL of eGFP RNA sample 1* (from RAD115) was combined with 20uL Phenol/Chloroform/Isoamyl alcohol </li>
<li>Shaken vigorously inside the fume hood for 15 seconds.</li>
<li>Centrifuged at 12000G at 4C for 2 min.</li>
<li>The top aqueous layer was transferred to a new epp and combined with a new 20uL of phenol/chloroform/isoamyl alcohol.</li>
<li>Shaken vigorously inside the fume hood for 15 seconds.</li>
<li>Centrifuged at 12000G at 4C for 2 min.</li>
<li>The aqueous layer was transferred to a new epp.</li>
<li>50uL of isopropanol was added and shaken.</li>
<li>Centrifuged at 20000G at 4C for 10min.</li>
<li>The isopropanol was removed and the pallet was washed with 40 uL of ice-cold isopropanol.</li>
<li>Centrifuged at 25000G at 4C for 5 min.</li>
<li>The isopropanol was removed and the palate was dried to air.</li>
<li>The pallet was resuspended in 10 uL milliQ. sample labelled: RNA pure GFP 18-07-24.</li>
<li>The concentration of the purified RNA was measured with nanodrop to be 2562 ng/uL.</li>
</ol>
<h4>Discussion:</h4>
<p>
The nanodrop result shows a promising result, the concentration of the purified RNA is high enough to continue with further experiments.
To be certain/to see that the purification worked well enough it is decided that the purified sample will be ran on a gel.
</p>
</td></tr>
<tr><td class="journal-td">
<p>22nd of July 2024</p>
<h3>1st Trial mRNA Purification, Gel Results.</h3>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
<li>Maks</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><p style="background-color:yellow">
No protocol or same protocol (??) <!-- Insert Protocol -->
</p></li>
</ul>
<h4>Experimental:</h4>
<ol>
<li>To prevent high concentrations of RNA on the gel, the 10 uL sample was diluted with 850uL MilliQ.</li>
<li>5uL of the diluted sample was combined with 15uL of denaturing RNA loading buffer, incubated at 65C for 5min.</li>
<li>The quality of the RNA was investigated by a denaturing agarose gel (110V, 50 min).</li>
</ol>
<h4>Discussion</h4>
<p>
The gel showed no bands or smears, possibly because the sample was too diluted.
A second trial for the purification needs to be done to try and understand this result.
</p>
</td></tr>
<tr><td class="journal-td">
<p>22nd of July 2024</p>
<h3>2nd Trial mRNA Purification.</h3>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
<li>Maks</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><p style="background-color:yellow">
No protocol (??) <!-- Insert Protocol -->
</p></li>
</ul>
<h4>Experimental:</h4>
<ol>
<li>We tried to reconcentrate the diluted RNA sample (see RAD116) by percipitating it with 200uL ice cold isopropanol and centrifuging at 27500 rfc at 4C for 5 min → did not do anything.</li>
<li>To the remaining 20 uL of sample 1* (see RAD115), 20 uL phenol/chlorophorm was added and shaken.</li>
<li>The sample was centrifuged at 12000 rfc, 4C for 2 min</li>
<li>The top (aqueous) layer was transported to a new epp together with 20 uL new phenol chlorophorm and shaken.</li>
<li>Centrifuged at 12000 rfc, 4C for 2 min.</li>
<li>Top layer was added to 50 uL ethanol, shaken.</li>
<li>Centrifuged at 29000 rfc, 4C for 10 min.</li>
<li>Supernatant was removed and the palete was washed with 40 uL ice cold ethanol.</li>
<li>Centrifuged at 29000 rfc, 4C for 5 min.</li>
<li>The supernatant was removed and the palete was air dried.</li>
<li>A small bit of the palete was removed, and resuspended in 10uL milliQ for analysis.</li>
<li>The rest of the palete is stored in the freezer, before use it should be resuspended in milliQ.</li>
<li>The fragment of the palete was analysed on a denaturating agarose gel (110V, 50min).</li>
<li>Absolutely no bands were observed on the gel.</li>
</ol>
<h4>Discussion</h4>
<p>
The problem is most likely not caused by a too dilute sample, but it could be the result of the buffer being multiple days old.
It might have picked up RNases that have degraded the sample while in the gel.
</p>
</td></tr>
<tr><td class="journal-td">
<p>23rd of July 2024</p>
<h3>2nd Trial mRNA Purification, Gel Results.</h3>
<h4>Participants:</h4>
<ul>
<li>Maks</li>
<li>Thijmen</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li><p style="background-color:yellow">
No protocol (??) <!-- Insert Protocol -->
</p></li>
</ul>
<h4>Experimental:</h4>
<ol>
<li>Fresh buffer was prepared</li>
<li>The palete was resuspended in 15uL milliQ and the concentration was measured to be 1881 ng/uL.</li>
<li>5uL of the sample was run on a denaturing agarose gel (110V 50min)</li>
<li>No bands were observed (in the picture the first column should have been the purified GFP, the other 2 bands are non purified F8 samples).</li>
</ol>
<img class="journal-img" src="https://static.igem.wiki/teams/5342/images/notebook/mrna/mrna-purification-1.webp" alt="mRNA Purification">
<h4>Discussion</h4>
<p>
The problems are not caused by a diluted sample or by an old buffer. It might be a more fundamental problem with the purification method.
We might need to look for a new way to do the purification.
</p>
</td></tr>
</table>
</div>
</div>
</div>
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<h2>Inspirations</h2>
<hr>
<ul>
<ul>
<li><a href="http://2018.igem.org/Team:Munich/Notebook">2018 Munich</a></li>
<li><a href="https://2019.igem.org/Team:Georgia_State/Notebook">2019 Georgia State</a></li>
<li><a href="https://2019.igem.org/Team:Newcastle/Notebook">2019 Newcastle</a></li>
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</ul>
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