<h2>RAD129: Cap-0 synthesis using Vaccinia Capping Enzyme (NEB #M2080)</h2>
<p>
<b>Background:</b> We used the following
<ahref="https://www.neb.com/en/protocols/0001/01/01/capping-protocol-m2080"target="_blank">capping protocol</a> for the following.
</p>
<p>Post-transcriptional capping and Cap-1 methylation</p>
<p>
Post-transcriptional capping is often performed using the mRNA capping enzyme from Vaccinia virus or Faustovirus.
This enzyme complex converts the 5´-triphosphate ends of in vitro transcripts to m7G-cap (Cap-0) required for efficient protein translation in eukaryotes.
comprises three enzymatic activities (RNA triphosphatase, guanylyltransferase, guanine N7-methyltransferase) that are necessary for the formation of the complete Cap-0 structure, m7Gppp5´N, using GTP and the methyl donor S-adenosylmethionine.
As an added option, the inclusion of the mRNA Cap 2´ O-Methyltransferase (NEB #M0366) in the same reaction results in formation of the Cap-1 structure (m7Gppp5´Nm), a natural modification in many eukaryotic mRNAs responsible for evading cellular innate immune response against foreign RNA.</p>
<p>This enzyme-based capping approach results in a high proportion of capped message, and it is easily scalable. The resulting capped RNA can be further modified by poly(A) addition before final purification.</p>
<p>This protocol is designed to cap up to 10 µg of RNA (100 nt or larger) in a 20 µl reaction. Reaction size can be scaled up, as needed. The system provides enough reagents to perform 40 reactions at the 10 µg RNA/20 µl reaction scale.</p>
<p>Samples:</p>
<olclass="emptylist">
<li>C1: 13.5 ul rna (A2d +C2d) + 1.5 ul MilliQ = 15 ul</li>
<li>C2: 7.5 ul rna (G2d) + 7.5 ul MilliQ = 15 ul</li>
<li>GFP: 6 ul rna (L5) + 9 ul MilliQ = 15 ul</li>
</ol>
<p>Aliquots of 2ul of SAM. +30 ul milliq = 32 ul of SAM (2 mmol) (Because SAM degrades very easily and for every sample, new aliquot was used)</p>
<p>For GFP: 13.3 ug = 1.33 times more than specified in protocol, so the volumes of materials for the GFP sample were increased.</p>
</td></tr>
<tr><tdclass="journal-td">
<tr><tdclass="journal-td">
<p>1st Jan 1970</p>
<p>26th of August 2024</p>
<h3>Example loaded in</h3>
<h4>Participants:</h4>
<h4>Participants:</h4>
<ul>
<ul>
<li>Tim</li>
<li>Henriete</li>
<li>Thijmen</li>
</ul>
</ul>
<h4>Protocols:</h4>
<h4>Experimental:</h4>
<ol>
<li>RNA and MilliQ in a 1.5 ml microfuge tube was added to a final volume of 15.0 µl.</li>
<li>Heated at 65°C for 5 minutes.</li>
<li>Samples were left on ice for 5 min.</li>
<li>
the following components were added in the order specified:
<ul>
<li>Denatured RNA (from above): 15.0 µl for F8 , 19.95 ul for GFP</li>
<li>10X Capping buffer: 2.0 µl for F8, 2.66ul for GFP</li>
<li>GTP (10 mM) :1.0 µl for F8, 1.33 ul for GFP</li>
<li>SAM (2 mM, dilute 32 mM stock to 2 mM): 1.0 µl for F8, 1.33ul for GFP</li>
<li>Vaccinia Capping Enzyme: 1.0 µl for F8, 1.33 ul GFP. Incubate at 37°C for 30 minutes.</li>
</ul>
</li>
<li>Incubate at 37°C for 30 minutes.</li>
<li>RNA is now capped and ready for use in downstream applications.</li>
</ol>
<ulclass="emptylist">
<li>NOTE: when adding the GTP, 1.33 ul was accidentally added to all samples. It technically should not change the proficiency of capping as it is the guanosine triphosphate.</li>
<li>NOTE: The samples were prepared until vaccina enzyme and left on ice for a certain time due to the heating block being too hot and took a while to cool down</li>
</ul>
<p>Purification of the capped samples:</p>
<ol>
<li>80 ul MilliQ and 100 ul of chloroform was added and shaken vigorously</li>
<li>samples were shaken vigorously in the fume hood for 15 seconds.</li>
<li>centrifuged at 12000 G at 4C for 2 min</li>
<li>the aqueous phase was transferred to another epp</li>
<li>10ul of 3 mol sodium acetate was added together with 300 ul ethanol</li>
<li>samples were shaken vigorously in the fume hood for 15 seconds.</li>
<li>centrifuged at 25000G at 4C for 10 min</li>
<li>50 ul of 70% ethanol was added</li>
<li>samples were shaken vigorously in the fume hood for 15 seconds.</li>
<li>centrifuged at 25000G at 4C for 5 min</li>
<li>Pellet was dried and 10 ul MilliQ was added.</li>
</ol>
<h4>Results and Discussion:</h4>
<p>As far as we can tell the capping was done successfully since we followed the provided protocol, however, we can’t check right now if it has been successful since we will only place the samples on the gel when the whole RNA modification is done. Tomorrow we will continue with the tailing so for now the samples are stored in the freezer (-20 degrees Celcius). </p>
<p><b>Conclusion:</b> The RNA capping has been done successfully, so we can proceed with tailing the samples tomorrow.</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD130: A-tailing using E. coli Poly(A) Polymerase</h2>
<p>
<b>Background:</b> The tailing protocol that we used is found
<p>The poly(A) tail confers stability to the mRNA and enhances translation efficiency. The poly(A) tail can be encoded in the DNA template by using an appropriately tailed PCR primer, or it can be added to the RNA by enzymatic treatment with E. coli Poly(A) Polymerase (NEB #M0276). The length of the added tail can be adjusted by titrating the Poly(A) Polymerase in the reaction (Figure 6).</p>
<p>The importance of the A-tail is demonstrated by transfection of untailed vs. tailed mRNA. When luciferase activity from cells transfected with equimolar amounts of tailed or untailed mRNAs were compared, a significant enhancement of translation efficiency was evident (Figure 6). HiScribe T7 ARCA mRNA Synthesis Kit (with tailing) (NEB #E2060) includes E. coli Poly(A) Polymerase, and enables a streamlined workflow for the enzymatic tailing of co-transcriptionally capped RNA.</p>
<p>GFP sample has 13.3 ug = 1.33 times more than required in protocol, so the volumes of materials for the GFP sample were increased. To make 1X E. coli Poly(A) Polymerase (5X) Reaction Buffer: 4ul E. coli Poly(A) Polymerase + 16 ul water =20 ul 1x E. coli Poly(A) Polymerase Reaction Buffer</p>
<p>Add the following components in the order specified:</p>
<ol>
<li>RNA + MilliQ to a total volume of 15 ul for F8 (19.95 ul for GFP)</li>
<li>5X E. coli Poly(A) Polymerase Reaction Buffer: 2 µl (1X) for F8 (2.66 ul for GFP)</li>
<li>ATP (10mM): 2 µl for F8 (2.66 ul for GFP)</li>
<li>E. coli Poly(A) Polymerase: 1 µl for F8 (1.33 ul for GFP)</li>
<li>Total volume 26.6 ul for GFP and 20 ul for F8</li>
<li>Incubate reaction at 37°C for 30 minutes.</li>
<li>Stop the reaction by adding EDTA to a final concentration of 10 mM or directly proceeding to the cleanup step.</li>
</ol>
<h4>Participants:</h4>
<ul>
<li>Henriete</li>
</ul>
<ol>
<li>80 ul (73.4 ul for GFP sample) MilliQ and 100 ul of chloroform was added and shaken vigorously</li>
<li>samples were shaken vigorously in the fume hood for 15 seconds.</li>
<li>centrifuged at 12000 G at 4C for 2 min</li>
<li>the aqueous phase was transferred to another epp</li>
<li>10ul of 3 mol sodium acetate was added together with 300 ul ethanol</li>
<li>samples were shaken vigorously in the fume hood for 15 seconds.</li>
<li>centrifuged at 25000G at 4C for 10 min</li>
<li>50 ul of 70% ethanol was added</li>
<li>samples were shaken vigorously in the fume hood for 15 seconds.</li>
<li>centrifuged at 25000G at 4C for 5 min</li>
<li>Pellet was dried and 10 ul MilliQ was added.</li>
</ol>
<p>Note: in step 5 only 70% ethanol was added instead of 100%. The 30% water might have dissolved the rna. The samples were centrifuged at 30000G for 5 min again. 100 ul 100% ethanol was added and samples were centrifuged for 5 min at 25000G.</p>
<h4>Participants:</h4>
<ul>
<li>Henriete</li>
</ul>
<ol>
<li>100 ml TBE 1x buffer : 90 ml Milliq + 10 ml TBE (10X)</li>
<li>50 ml buffer + 0.35g agarose + 2.5 ul EtBr.</li>
<p>Figure 1. gel electrophoresis of modified mRNA (well 11: modified F8 mRNA 1, 12: modified F8 mRNA 2, 13: modified GFP mRNA, 14: positive control) samples to the left belong to RAD131. </p>
<p>
<p>
Procedure:
Only the modified GFP mRNA is visible on this gel. Suggesting that capping and tailing of this RNA was successful, but that of the F8 RNA was not. This could be because the F8 RNA was already less stable.
</p>
<p><b>Conclusion:</b> The modification was successful on the GFP RNA, but the F8 RNA should be repeated. To get around our problems with purifying the F8 RNA, Frank tried our method.</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD132: Capping and tailing of GFP (sample G) and F8 mRNA</h2>
<p>
<b>Background:</b> In this experiment, a second attempt at tailing and capping is done. In the last experiments (RAD129-RAD130), a small amount of GFP RNA was successfully modified. modification of F8 RNA was not yet successful, but we received a large amount of purified F8 RNA from our supervisor Frank which might have a better quality and will hopefully stay intact during the tailing and capping.
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>4th of September 2024</p>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
<li>Thijmen</li>
</ul>
<p>Capping:</p>
<p>
a new 2 mM SAM dilution was prepared: 2uL (32mM)SAM + 30uL MilliQ.
Sample prep:
</p>
</p>
<ol>
<ol>
<li>Put example in</li>
<li>3.3 uL of sample G was used to get 10ug and diluted with MilliQ to a final volume of 15uL</li>
<li>See if something works</li>
<li>3.6 ul F8 RNA was used to get 10ug and diluted with MilliQ to a final volume of 15uL</li>
<li>Forget to remove it</li>
<li>Heated at 65C for 5 min</li>
<li>placed on ice for 5 min</li>
<li>
the following components were added in the order specified:
<ul>
<li>10X Capping buffer: 2.0 µl</li>
<li>GTP (10 mM):1.0 µl</li>
<li>SAM (2 mM, dilute 32 mM stock to 2 mM): 1.0 µL</li>
<li>Vaccinia Capping Enzyme: 1.0 µl.</li>
</ul>
</li>
<li>Incubate at 37°C for 30 minutes. (left the sample on ice for a bit while waiting for the heating block to cool down</li>
</ol>
<p>purification: (same protocol as in RAD129)</p>
<ol>
<li>80 ul MilliQ and 100 ul of chloroform was added to the sample and shaken vigorously in the fumehood for 15 seconds</li>
<li>centrifuged at 12000 G at 4C for 2 min</li>
<li>the aqueous phase was transferred to another epp</li>
<li>10ul of 3 mol sodium acetate was added together with 300 ul ethanol. samples were shaken vigorously in the fume hood for 15 seconds.</li>
<li>centrifuged at 25000G at 4C for 10 min</li>
<li>supernatant was removed and discarded</li>
<li>50 ul of 70% ethanol was added</li>
<li>samples were shaken vigorously in the fume hood for 15 seconds.</li>
<li>centrifuged at 25000G at 4C for 5 min</li>
<li>supernatant was removed and the pellet was dried to the air</li>
<li>pellet was resuspended in 15ul milliQ</li>
</ol>
</ol>
</td></tr>
</td></tr>
<tr><tdclass="journal-td">
<tr><tdclass="journal-td">
<p>2nd Jan 1970</p>
<p>5th of September 2024</p>
<h3>Example getting removed</h3>
<h4>Participants:</h4>
<h4>Participants:</h4>
<ul>
<ul>
<li>Tim</li>
<li>Lea</li>
<li>Thijmen</li>
</ul>
</ul>
<h4>Protocols:</h4>
<p>tailing: (same protocol as RAD130)</p>
<ol>
<li>
reaction mixtures for both samples were prepared by adding the following components:
<ul>
<li>RNA: purified sample</li>
<li>4ul 5X E. coli Poly(A) Polymerase Reaction Buffer</li>
<li>2ul ATP (10mM)</li>
<li>1ul E. coli Poly(A) Polymerase</li>
</ul>
</li>
<li>Incubate at 37°C for 30 minutes</li>
<li>purification:(same protocol as above)</li>
<li>NOTE: a different centrifuge was used with a max RPM of 20000 instead of 25000</li>
</ol>
</td></tr>
<tr><tdclass="journal-td">
<p>6th of September 2024</p>
<h4>Participants:</h4>
<ul>
<ul>
<li>Undoing my mistakes protocol</li>
<li>Maks</li>
<li>Thijmen</li>
</ul>
</ul>
<h4>Experimental:</h4>
<p>analysis: </p>
<p>The two samples were analyzed on a 0.7% 50 min 110V</p>
<p>Figure 1. After capping and tailing the gel showed no stain</p>
<p>
<p>
Procedure:
Even though the capping went well (pellet was seen), the tailing went bad (no pellet, even though the pellet should become even bigger). This might be due to absence of Mg2+ in the tailing enzyme.
</p>
</p>
<p><b>Conclusion:</b> The capping procedure from the experiment RAD 132 worked out. However, after the tailing procedure no product was seen. The gel showed no stain. The Mg2+ will be added to the tailing enzyme.</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD133: Capping and tailing of GFP (sample G) and F8 mRNA</h2>
<p>
<b>Background:</b> The capping procedure from the experiment RAD 132 worked out. However, after the tailing procedure no product was seen. The gel showed no stain. No MnCl2 was added in the last experiment, this would have inhibited the poly(A) formation. However, it does not explain the lack of yield. In this experiment, the procedure is repeated with minor adjustments and with
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>11th of September 2024</p>
<h4>Participants:</h4>
<ul>
<li>Kate</li>
<li>Lea</li>
<li>Thijmen</li>
</ul>
<h4>Experimental:</h4>
<ulclass="emptylist">
<li>2 mM SAM dilution was prepared: 2uL (32mM)SAM + 30uL MilliQ </li>
<li>8 samples were prepared (4GFP & 4 F8):</li>
<li>F8: 3.6 ul F8 RNA was used to get 10ug and diluted with MilliQ to a final volume of 15uL (4x)</li>
<li>GFP(1): 3.3 uL of sample G was used to get 10ug and diluted with MilliQ to a final volume of 15uL</li>
<li>GFP(4): 6.68 ul GFP from RAD120 1 to get 10ug and diluted with MilliQ to a final volume of 15uL </li>
<li>GFP(2,3): 5.47 ul GFP from RAD120 2 to get 10ug and diluted with MilliQ to a final volume of 15uL </li>
<li>0.7% Agarose gel electrophoresis of denatured mRNA samples: </li>
<li>1st, 2nd, 3rd, 9th row shows a smear (F8), 11th row shows a band (GFP form Frank)</li>
<li>Other lanes are empty</li>
<li>Reason assumed: concentrations of the sample are too low to be visible.</li>
</ul>
<p>Nanodrop results:</p>
<tableclass="journal-table">
<tr>
<td>F8 1</td>
<td>417.6 ng/ul</td>
</tr>
<tr>
<td>F8 2</td>
<td>284.3 ng/ul</td>
</tr>
<tr>
<td>F8 3</td>
<td>464.4 ng/ul</td>
</tr>
<tr>
<td>F8 4</td>
<td>586.4 ng/ul</td>
</tr>
<tr>
<td>GFP 1</td>
<td>501.5 ng/ul</td>
</tr>
<tr>
<td>GFP 2</td>
<td>767.2 ng/ul</td>
</tr>
<tr>
<td>GFP 3</td>
<td>426 ng/ul</td>
</tr>
<tr>
<td>GFP 4</td>
<td>848.8 ng/ul</td>
</tr>
</table>
<p><b>Conclusion:</b> The concentration of RNA was too low to be seen on the gel, but due to the time constraints, it was decided to move on with this RNA.</p>
