<b>Background:</b> The purpose of the experiment is to make the transfected bacteria with an inserted FVIII vector plasmid.
The bacteria are cultured on antibiotic full media, so only the ones that have absorbed this plasmid can survive.
The bacterial cultures used are Top10 and XL1Blue (both E.coli strains optimized for plasmid amplification)
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>1st Jan 1970</p>
<h3>Example loaded in</h3>
<p>3rd of July 2024 - 4th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Tim</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li>Testing protocol</li>
<li>Lea</li>
</ul>
<h4>Experimental:</h4>
<p>
Procedure:
</p>
<p>Procedure:</p>
<ol>
<li>Put example in</li>
<li>See if something works</li>
<li>Forget to remove it</li>
<li>Top10Transf colony was prepared the following way: 1ul FVIII plasmid solution was mixed with 200ul Top10 bacteria suspension.</li>
<li>XL1BlueTransf colony was prepared the following way: 1ul FVIII plasmid solution was mixed with 200ul XL1Blue bacteria suspension.</li>
<li>Both of the cultures were left on ice for 0.5 h.</li>
<li>Both bacteria were heat shocked for 54 seconds at 42C.</li>
<li>800 ul of LB medium.</li>
<li>Left in shaker for 0.5h 37C.</li>
<li>Bacteria were spun down with a centrifuge and 900uL medium was removed</li>
<li>The pallet was resuspended in the remaining 100uL medium and plated on agar plates containing ampicillin.</li>
<li>The bacteria were left in the incubator (37 C)overnight.</li>
<li>(the next day)</li>
<li>after 24 h incubation, the Top10 plate had many miniscule colonies, while Xl1Blue had less, but larger colonies.</li>
<li>6 colonies were picked from the XL1Blue plate and 4 from the Top10 plate.</li>
<li>in total 10 5mL liquid cultures were prepared and incubated overnight at 37C.</li>
</ol>
<p><b>Conclusion:</b> These successful bacteria synthesis allows for further FVIII isolation and production (RAD110)</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD110: Miniprep of cp FVIII DNA and digestion</h2>
<p>
<b>Background:</b> Extraction of the DNA samples from the bacteria cultures, prepared in the RAD107 experiment: Top10 and XL1 Blue.
Plasmid used is linked <ahref="https://www.addgene.org/41036/">here</a>.
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>2nd Jan 1970</p>
<h3>Example getting removed</h3>
<p>5th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Tim</li>
<li>Kate</li>
</ul>
<h4>Protocols:</h4>
<h4>Experimental:</h4>
<p>Cultures used: </p>
<ul>
<li>Undoing my mistakes protocol</li>
<li>E. Coli XL1 Blue: 1, 2, 3, 4, 5, 6 cultures with pc DNA Factor VIII</li>
<li>E. Coli: Top 10 1, 2, 3, 4 cultures with pc DNA Factor VIII</li>
</ul>
<h4>Experimental:</h4>
<p>Procedure:</p>
<ol>
<li>2ml of each bacterial culture was added. Labeled respectively: XL1, XL2, XL3, XL4, XL5, XL6; Top1, Top2, Top3, Top4</li>
<li>I centrifuged the pipetted cultures for 3 minutes at 1000 rpm, RT</li>
<li>Supernatant was collected leaving pellets dry</li>
<li>2 ml more of of cultures were added to each sample</li>
<li>Samples were centrifuged again, same way</li>
<li>Supernatant was collected leaving pellets dry</li>
<li>250 ul of P1 buffer was added to all samples</li>
<li>To fractions XL1-5 250 ul of P2 buffer were added</li>
<li>Samples XL1-5 were inverted 6 times (kept in the buffer P2 less than 5 min)</li>
<li>350 ul of N3 buffer was added to solutions XL1-5, samples were rotated 4-6 times [NOTE: XL2 had too much liquid (~1.25 ml), but pH paper showed that it is okay, so probably N3 buffer was added 2 times)]</li>
<li>Same as to XL1-5 was doe to XL6 and Top1-4</li>
<li>The samples were put to centrifuge 10 min at 13000 rpm, 20C</li>
<li>800 ul supernatants was collected to QIAprep spin column</li>
<li>This supernatant in QIA prep was centrifuged for 30-60 seconds at 13000 rpm, and the flow-though was discarded</li>
<li>250 ul PE buffer were added</li>
<li>Centrifuged 60 sec</li>
<li>flow-through was discarded</li>
<li>250 ul PE buffer were added</li>
<li>Centrifuged 60 sec</li>
<li>flow-through was discarded</li>
<li>50 ul H2O MiliQ added</li>
<li>Centrifuged 60 sec</li>
<li>flow-through was discarded</li>
<li>50 ul H2O MiliQ added</li>
</ol>
<p>Digestion:</p>
<ul>
<li>Combine EcoRI with plasmid for 10 digestions</li>
<li>Combine XhoI with plasmid for 10 digestions</li>
<li>From pc DNA FVIII (12086 bp)</li>
<li>XL1 Blue: 2.5 ul to EcoRI and 2.5 ul to XhoI & Top10 2.5 ul to EcoRI and 2.5 ul to XhoI</li>
</ul>
<p>Linearisation:</p>
<ul>
<li>EcoRI: 4 expected bands to be seen on gel: cuts at 347bp, 1728bp, 4688bp, 5323bp</li>
<li>XhoI: 1 expected bands to be seen on gel: cuts at 12.1 kbp</li>
<li>Composition for linearization mixture per one linearization: 2.5 ul buffer, 0.5 ul enzyme, 5 ul DNA, 17 ul MQ</li>
<li>Sample with enzymes is linearized at 37 C for 2 hours</li>
<b>Conclusion:</b> FVIII pcDNA was extracted from the bacterial cultures Top10 and XLBlue.
The gel analysis of enzymatically linearized DNA confirmed its proper structure.
The further steps of the FVIII DNA processing is the linearization of more plasmid, and then purifying it.
This DNA then should be transcribed to FVIII mRNA.
</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD111: Large Xho1 digestion</h2>
<p>
<b>Background:</b> Scaled up linearisation of plasmids for the use in in vitro transcription.
The XL1Blue plasmids are used because they were isolated in a higher concentration.
The Xho1 digestion will linearise the plasmid right after the coding sequence, allowing for run off transcription.
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>8th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
</ul>
<h4>Experimental:</h4>
<p>Cultures used: </p>
<ul>
<li>E. Coli XL1 Blue: 1, 2, 3, 4, 5, 6 cultures with pc DNA Factor VIII</li>
<li>E. Coli: Top 10 1, 2, 3, 4 cultures with pc DNA Factor VIII</li>
</ul>
<p>Procedure:</p>
<ol>
<li>Realise you forgot to remove example</li>
<li>Remove it</li>
<li>Commit, push, merge</li>
<li>the concentration of all isolated plasmid samples was determined</li>
<li>4 enzyme digestions with a volume of 150 uL were prepared using plasmids isolated from XL1Blue following the protocol. The DNA concentration of the plasmids used was 758 and 756 ng/uL,so 11.9 uL was used to reach 9ng pcDNA.</li>
<li>digestion was incubated at 37 C for 3 hours</li>
<li>The digested plasmids were purified using a spin column (following the same protocol as before):</li>
<li>600uL PB buffer to 120uL digestion mix, centrifuge for 1 min at 13000 rpm</li>
<li>the concentrations of the purified product were measured to be 79.9, 96.6, 94.7 and 88.8 ng/uL in that order</li>
<li>the purified products were put on gel (110V 50 min) to check the quality</li>
<li>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)</li>
<li>samples were loaded: ladder-unpurified———middle wells empty———1-2-ladder-3-4</li>
<p>The digested plasmids show clear bands at the length where we expected them. </p>
<p>
<b>Conclusion:</b> A large amount of linear FVIII pcDNA was produced and purified. This can now be used to perform in vitro transcription and produce FVIII mRNA.
</p>
</td></tr>
</table>
</div></div>
<divid="page-IVT"class="retrievable">
<pid="IVT-task">
<!-- Insert the name of the notebook page riight there. -->
Name of Notebook Task/Page
IVT and mRNA Purification
</p>
<pid="IVT-datetime">
<!-- Insert the time(s) below this comment-->
01/01/1970 - 21/12/12
09/07/24 - 30/08/24
</p>
<ulid="IVT-participants">
...
...
@@ -1213,61 +1308,52 @@
<li>Participant n</li>
</ul>
<ulid="IVT-protocols">
<!-- Insert each protocol within its own <li></li> tag below.-->
<h2>RAD112: IVT of the GFP and Factor VIII RNA</h2>
<p>
Procedure:
<b>Background:</b> The “In vitro synthesis of RNA using T7 RNA polymerase” protocol was used to translate mRNA from the cpFVIII DNA samples received in the experiment RAD110, as well as the GFP DNA received in the experiment RAD109.
</p>
<ol>
<li>Put example in</li>
<li>See if something works</li>
<li>Forget to remove it</li>
</ol>
</td></tr>
<tr><tdclass="journal-td">
<p>2nd Jan 1970</p>
<h3>Example getting removed</h3>
<p>9th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Tim</li>
</ul>
<h4>Protocols:</h4>
<ul>
<li>Undoing my mistakes protocol</li>
<li>Lea</li>
</ul>
<h4>Experimental:</h4>
<p>
Procedure:
</p>
<ol>
<li>Realise you forgot to remove example</li>
<li>Remove it</li>
<li>Commit, push, merge</li>
<li>a mastermix for 10 reactions of 20uL was prepared following the protocol for IVT. → ! Guanosine-5’-monophosphate was not used in the reaction mix, because it prohibits capping. mastermix amounts:</li>
<li>prepare 10 epps, with each 20uL mastermix</li>
<li>1uL dsDNA of the five different DNA samples (4 FVIII DNA, 1 GFP DNA) was added to epps (for FVIII linearised plasmid, 75 ng of DNA is needed for 20uL reaction, for GFP it is 26ng. The concentration of the FVIII DNA was close enough that 1uL could be taken from the tube. The GFP DNA was diluted 1:1 to a concentration of 30ng/uL and 1uL was used from that )</li>
<li>For every different DNA sample, one epp got 2uL T7 polymerase and the other 4uL. → 2uL is the amount recommended by the protocol, but because of the length of the FVIII gene, we wanted to see if a larger amount of T7 would be beneficial.</li>
<li>The reaction was incubated at 37 C for 3 hours.</li>
<li>5uL of all samples was mixed with 2uL loading dye and run on an agarose gel (110V 40 min)</li>
<p>Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.</p>
<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.
The FVIII RNA was expected to be longer than 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. maybe the calculations on the amount of template needed was incorrect.
</p>
<p>
→ I (Lea) recalculated the amount of template needed and I did receive a different outcome: 45 ng/20uL. This is a 15ng difference from what was used in this experiment.
</p>
<p><b>Conclusion:</b> Experiment needs to be repeated with proposed changes:</p>
<ul>
<li>different version of T7 poly.</li>
<li>Add RNAse inhibitor.</li>
<li>Reduce temperature to prevent autocatalytic breakdown.</li>
</ul>
</td></tr>
</table>
</div></div>
<divid="page-Purification"class="retrievable">
<pid="Purification-task">
<!-- Insert the name of the notebook page riight there. -->
