<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>
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:
<ul>
<li>Tris (1M): 8uL</li>
<li>MgCl2 (1M): 5uL</li>
<li>DTT(0.1M): 10uL</li>
<li>spermidine (25mM): 8uL</li>
<li>rNTPs(20mM): 40uL</li>
<li>MilliQ: 129uL</li>
<li>total: 200uL</li>
</ul>
</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>
...
...
@@ -1334,7 +1350,7 @@
<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.
...
...
@@ -1351,6 +1367,563 @@
<li>Reduce temperature to prevent autocatalytic breakdown.</li>
</ul>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD113: 2nd IVT iteration </h2>
<p>
<b>Background:</b> Mutated T7 (polymerase) is the T7 used last time, now we are also testing the wild type.
We are redoing the EGFP with the new calculation and trying an RNAse inhibitor to try and stop RNA degradation.
</p>
<p>Calculations:</p>
<tableclass="borderless-true">
<tr>
<td>EFGP + mutT7 = 9</td>
<td>EGFP + mutT7 + RNAse inh. = 10</td>
<td>EGFP + WT T7 = 4</td>
<td>EGFP + WT T7 + RNAse inh. = 5</td>
</tr>
<tr>
<td></td>
<td>F8 -1 + mut T7 + RNAse inh. = 3</td>
<td>F8 - 1 + WT T7 = 1</td>
<td>F8 - 1 + WT T7 + RNAse inh. = 2</td>
</tr>
<tr>
<td></td>
<td>F8- 4 +mut T7 + RNAse inh. = 8</td>
<td>F8 - 4 + WT T7 = 6</td>
<td>F8 - 4 + WT T7 + RNAse inh. = 7</td>
</tr>
</table>
</td></tr>
<tr><tdclass="journal-td">
<p>10th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
<li>Thijmen</li>
</ul>
<h4>Experimental:</h4>
<p>IVT:</p>
<ol>
<li>a 200 uL mastermix was prepared the same as the one in RAD112</li>
<li>The mix was divided over 10 epps labeled the same as the descriptions in the table.</li>
<li>
samples were prepared following the table:
<ul>
<li>4uL of T7 was used for both versions</li>
<li>0.5 uL of RiboLock RNase inhibitor was used</li>
<li>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.</li>
</ul>
</li>
<li>The reactions were incubated for 3 hours.</li>
</ol>
<p>Gel electrophoresis:</p>
<ol>
<li>Samples with dye were prepared - 5 ul sample + 2 ul dye.</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>
During the pipetting of the sample 3 the pipette tip fell off, which may be the cause of the absent band.
Samples 4 and 5 seem pure with only minor smearing.
Samples 2 and 7 have a significant smear, which could be perhaps resolved by using denaturing conditions that would eliminate the RNA’s autocatalytic activity.
</p>
<p>
<b>Conclusion:</b> Adding an RNase inhibitor improves the outcome of IVT.
Sample analysis on denaturing gel should be carried out in order to eliminate autocatalytic activity of the RNA molecule.
GFP samples give more clear bands on the native gel than the factor VIII samples.
</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD114: IVT of GFP + FVIII</h2>
<p>
<b>Background:</b> For the IVT, the In vitro synthesis of RNA using T7 RNA polymerase protocol was used.
For IVT we need between 5 and 50 nM of dsDNA. We want 10 nM, for FVIII linearised plasmid, 75ng of DNA is needed for 20uL reaction, for GFP it is 26ng.
The concentrations 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 (from RAD112).
</p>
<p>Calculations:</p>
<tableclass="borderless-true">
<tr>
<td>EFGP + mutT7 = 9</td>
<td>EGFP + mutT7 + RNAse inh. = 10</td>
<td>EGFP + WT T7 = 4</td>
<td>EGFP + WT T7 + RNAse inh. = 5</td>
</tr>
<tr>
<td></td>
<td>F8 -1 + mut T7 + RNAse inh. = 3</td>
<td>F8 - 1 + WT T7 = 1</td>
<td>F8 - 1 + WT T7 + RNAse inh. = 2</td>
</tr>
<tr>
<td></td>
<td>F8- 4 +mut T7 + RNAse inh. = 8</td>
<td>F8 - 4 + WT T7 = 6</td>
<td>F8 - 4 + WT T7 + RNAse inh. = 7</td>
</tr>
</table>
</td></tr>
<tr><tdclass="journal-td">
<p>15th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Adrian</li>
<li>Thijmen</li>
</ul>
<h4>Experimental:</h4>
<ol>
<li>
a mastermix for 5 reactions of 40uL was prepared following the protocol for IVT.
→ ! Guanosine-5’-monophosphate was not used in the reaction mix, because it prohibits capping. mastermix amounts:
<ul>
<li>Tris (1M): 8uL</li>
<li>MgCl2 (1M): 5uL</li>
<li>DTT(0.1M): 10uL</li>
<li>spermidine (25mM): 8uL</li>
<li>rNTPs(20mM): 40uL</li>
<li>MilliQ: 129uL</li>
<li>total: 200uL</li>
</ul>
</li>
<li>prepare 5 epps, with each 40uL mastermix, labeled with numbers 1 - 5. Note - epp number 5 had an air droplet in the pipette tip, so it's doubtful whether it has the correct amount</li>
<li>Factor 8 DNA - 2, Factor 8 DNA 3, GFP diluted were measured on nanodrop for concentration:</li>
The following were added to epps 1 - 5 in order from left to right:
<ul>
<li>1 = 1.78 ul Factor 8 - 2 + 8 ul polymerase</li>
<li>2 = 1.78 ul Factor 8 - 3 + 8 ul polymerase</li>
<li>3 = 1.78 ul Factor 8 - 2 + 1 ul RNase inhibitor + 8 ul polymerase</li>
<li>4 = 1.78 ul Factor 8 - 3 + 1 ul RNase inhibitor + 8ul polymerase</li>
<li>5 = 2.14 ul GFP DNA + 1 ul RNase inhibitor + 8 ul polymerase</li>
</ul>
<p>
Note: From calculating the concentration of DNA specified in the manual (10nM) considering that our DNA is roughly 8000 bp which are all roughly 618g/mol each we calculated that the final concentration that manual uses is 49 ng/ul which is way higher than what we have used in all experiments so far.
</p>
<p>Note: Use the mutated polymerase frank gave us (green label)</p>
<p>Solutions transferred to PCR tubes labeled 1-5 with roman numbers</p>
<p>Solutions stored overnight in PCR machine at 4 degrees centigrade</p>
</li>
<li>
All 5 products analyzed on a denaturing agarose gel:
<ul>
<li>Added 0.35g agarose in 5 ml 10xMOPS solution and 36 mL MilliQ</li>
<li>Heat in microwave for about 15 seconds (until complete dissolution)</li>
<li>Stir continuously while slowly adding 9ml 37% formaldehyde</li>
<li>Stir for 5 seconds more and pour in casting tray</li>
The bands all show up at the same hight, while a significant difference in length was expected. the first 3 samples were made with GFP template DNA and the last 2 with F8, so the first 3 should be way lower compared to the last 2.
Although we were quite confident in our pipetting, this could be due to a pipetting mistake while adding the template DNA or when putting the samples on the gel.
RiboLock RNase inhibitor should always be addded in this procedure.
</p>
<p><b>Conclusion:</b> Only GFP mRNA was rececived & analysed on the gel. Samples 1*, 2*, 3*, 4*, 5*.</p>
<p><b>Next steps:</b></p>
<p>purification with phenol chlorophorm extraction.</p>
<p>optimise for GFP, then move on to FVIII.</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD116: 1st trial RNA purification using phenol:chloroform</h2>
<p>
<b>Background:</b> protocol based on:
<ahref="https://www.protocols.io/view/rna-clean-up-by-phenol-chloroform-yxmvmw46g3pe/v2?step=null">RNA Clean-up by phenol:chloroform v2</a>.
With the mRNA purification, we are trying to get samples which would only contain the mRNA that we want, this will be done by following a protocol which will lead to the removal of any IVT ‘ingredients’ that are present in the sample.
By removing these and keeping the mRNA <!-- unfinished sentence -->
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>18th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Adrian</li>
<li>Lea</li>
<li>Maks</li>
<li>Thijmen</li>
</ul>
<h4>Experimental:</h4>
<ol>
<li>20uL of eGFP RNA sample 1* (from RAD115) was combined with 20uL Phenol/Chloroform/Isoamyl alcohol (https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1)</li>
<li>shaken vigorously inside 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 fume hood for 15 seconds</li>
<li>centrifuged at 12000G at 4C for 2 min</li>
<li>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 pellet was washed with 40 uL 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 pellet was resuspended in 10 uL milliQ. sample labeled: 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>
<p>22nd of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
<li>Maks</li>
</ul>
<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>
<p>the gel showed nothing. possibly because the sample was too diluted.</p>
<h4>Results and Discussion:</h4>
<ul>
<li>Gel on purified product showed no product; problem not in running buffer.</li>
<li>Maybe, a mistake was made (someone pipetted the wrong fraction), maybe, the sample did not have the mRNA - only single nucleotides.</li>
<li>Should try to run the gel for 5 minutes.</li>
<li>Maybe, the reason is that mRNA doesn’t have a cap, it might denature. RNAse might break it down. </li>
<li>Maybe, we should first modify mRNA, and then purify. - look it up/ ask Frank. 3’-5’ mRNA</li>
</ul>
<p>Endo or exonuclease which might affect the mRNA.</p>
<ul>
<li>Before that - try to work with chemicals from Frank’s lab.</li>
<li>Maybe, Afka can help you to troubleshoot this purification. (Maks)</li>
</ul>
<p><b>Conclusion:</b>The purification procedure does not seem to yield the desired results. Procedure optimisation is required prior to further attempts. </p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD117: 2nd trial purification</h2>
<p>
<b>Background:</b> Further attempts of the purification of the IVT samples
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>22nd of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
<li>Maks</li>
</ul>
<h4>Experimental:</h4>
<ol>
<li>we tried to reconcentrate the diluted RNA sample (see RAD116) by precipitating 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/chloroform 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 chloroform 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 pellet 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 pellet was air dried.</li>
<li>a small bit of the pellet was removed, and resuspended in 10uL milliQ for analysis</li>
<li>the rest of the pellet is stored in the freezer, before use it should be resuspended in milliQ</li>
<li>The fragment of the pellet was analyzed 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>
<p>23rd of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Maks</li>
<li>Thijmen</li>
</ul>
<ol>
<li>Fresh buffer was prepaired</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>
<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. </p>
<p><b>Conclusion:</b> Optimization of the purification procedure is needed before the continuation of the experiments as the current procedure seems to yield suboptimal results.</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD118: repeat upscale IVT FVIII RNA</h2>
<p>
<b>Background:</b> Preparation of the new F8 mRNA as the previous samples have been stored in the freezer for a long time
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>22nd of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
<li>Maks</li>
</ul>
<h4>Experimental:</h4>
<ol>
<li>200 uL mastermix was prepared, mixed with 10 uL FVIII - 2 template DNA. the IVT mix was divided over 2 PCR epps and after incubating for 10 minutes, we realized we forgot to add T7</li>
<li>5 uL of WT T7 was added to each PCR tube.</li>
<li>the reaction was run in the PCR machine at a constant 37C for 3 hours, then kept at 4C overnight.</li>
</ol>
<p>23rd of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Maks</li>
<li>Thijmen</li>
</ul>
<ol>
<li>The IVT reactions were removed from the PCR machine and the RNA concentration was measured.</li>
<li>The tube with the red marking had a concentration of 7446ng/uL and the one without marking had a concentration of -6389ng/uL even after cleaning and re-blanking.</li>
<li>The two samples were run on a denaturing agarose gel (110V 50 min)</li>
</ol>
<h4>Results and Discussion:</h4>
<p>
The gel showed no bands, likely indicating either a complete samples’ degradation or a mistake made during the purification.
The latter idea is supported by the seemingly negative RNA concentration in the sample
</p>
<p><b>Conclusion:</b> New mRNA samples have to be prepared, care should be taken to avoid any potential mistakes during IVT setup or mRNA purification.</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD119: 5-minute gel test</h2>
<p>
<b>Background:</b> Previous experiments with purification led to results that were not favourable. This checkup is done in order to check whether there truly is no mRNA.
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>29th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
</ul>
<h4>Experimental:</h4>
<p>
The purified GFP mRNA sample was taken out of the fridge after which it was quickly vortexed.
In order to have enough sample to be able to pipet it 5ul of miliQ water was added to the sample.
After this 5uL of the purified mRNA GFP sample was added to an epp and 2ul of loading dye was added to the same epp.
This epp was then vortexed to mix the 2 ingredients.
Next, the sample + loading dye mixture was pipetted into the gel after which the gel was run for 5 minutes at 110 volts.
<p>Figure 1. Attempt 1 gel of purified GFP mRNA. 5 minute gel check.</p>
<h4>Discussion</h4>
<p>
As can be seen on the photo, even after 5 minutes of running the gel there is no band visible.
This would indicate that the current purification method is not working properly.
A new purification method is used in RAD120.
</p>
<p><b>Conclusion:</b> Purification method should be revised, and the new method repeated and performed.</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD120: Purification of GFP mRNA trial 3</h2>
<p>
<b>Background:</b> GFP samples 2* and 3* from the experiment RAD115.
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>29th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Maks</li>
<li>Thijmen</li>
</ul>
<p>Note: all the materials that were used during the purification (like epps and pipet points) were sterile and all the work was done with gloves. This all will ensure a RNase free environment.</p>
<ol>
<li>20uL of a GFP mRNA sample 1 was combined with 20uL <ahref="https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1"target="_blank">Phenol/Chloroform/Isoamyl alcohol</a></li>
<li>shaken vigorously inside the fume hood for 15 seconds.</li>
<li>centrifuged at 12000 G 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 fume hood for 15 seconds</li>
<li>centrifuged at 12000 G at 4C for 2 min</li>
<li>aqueous layer was transferred to a new epp</li>
<li>50uL of 96% ethanol was added and shaken</li>
<li>centrifuged at 25000G at 4C for 2min (this was a mistake and should have been 10 minutes)</li>
<li>The ethanol was removed and 50 ul of 70% ethanol solution was added to the sample.</li>
<li>centrifuged at 25000G at 4C for 5 min</li>
<li>The ethanol was removed and the palate was dried to air.</li>
<li>the pellet was resuspended in 10 uL milliQ. sample labeled: Pure GFP 29/07/2024</li>
</ol>
<p>29th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Maks</li>
<li>Thijmen</li>
</ul>
<p>
The purified GFP mRNA sample named Pure GFP 29/07/2024 was taken.
After this 5uL of the purified GFP sample was added to an epp and 2ul of loading dye was added to the same epp.
This epp was then vortexed to mix the 2 ingredients. Next, the sample + loading dye mixture was pipetted into the gel after which the gel was run for 5 minutes at 110 volts.
</p>
<p>30th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Thijmen</li>
</ul>
<p>
The purified GFP mRNA sample named Pure GFP 29/07/2024 was taken.
After this 5uL of the purified GFP sample was added to an epp containing 2ul of loading dye.
This epp was then vortexed to mix the 2 ingredients. Next, the gel was loaded.
In the first well 5 ul of a molecular ladder was pipetted and in the second well 5 ul of the sample + loading dye was pipetted. After this the gel was run for 50 minutes at 110 volts.
</p>
<p>30th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Thijmen</li>
</ul>
<p>A new gel was made by using the following protocol:</p>
<ol>
<li>100 ml of 0.5 xTBE was put in a flask</li>
<li>0.7 g of agarose was dissolved in the 100ml of 0.5 xTBE flask.</li>
<li>The solution was then mixed</li>
<li>The mixture with the agarose was heated in the microwave until it became clear</li>
<li>5 ul ethidium bromide was added to the mixture.</li>
<li>The gel was poured into the tray and solidified by waiting.</li>
</ol>
<p>
After the new gel was made the sample was prepared.
The purified GFP mRNA sample named Pure GFP 29/07/2024 was taken.
After this 5uL of the purified GFP sample was added to an epp containing 2ul of loading dye.
This epp was then vortexed to mix the 2 ingredients. Next, the gel was loaded.
In the first well 5 ul of a molecular ladder was pipetted and in the second well 5 ul of the sample + loading dye was pipetted.
After this the gel was run for 50 minutes at 110 volts.
</p>
<h4>Results and Discussion:</h4>
<p>
We hope that by replacing the isopropanol with ethanol and by working with sterile equipment that the mRNA will survive the purification step and that we will receive pure GFP mRNA.
In order to check if the purification really worked we have to run the samples on a gel.
<p>Figure 2. Gel electrophoresis of pure GFP RNA sample part 1. Date: 30-07-2024</p>
<p>
As you can see on the photo the sample looks pure as far as we can see.
However both the sample and the ladder are very vague.
This could be due to an error, a too low concentration (which would be bad in the case of the sample) or that the gel is too old.
It was hypothesized that the gel is too old since it has been made 1 or 2 weeks prior.
Next step is to refresh the gel and rerun the sample.
</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD121: Purification of mRNA trial 4, scaling upA</h2>
<p>
<b>Background:</b> Same phenol-chloroform procedure as in the RAD120 extraction with larger quantities of GFP and FVIII mRNA.
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>1st of August 2024</p>
<h4>Participants:</h4>
<ul>
<li>Maks</li>
</ul>
<h4>Experimental:</h4>
<ol>
<li>40 uL of a GFP mRNA sample 1 was combined with 40 uL <ahref="https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1"target="_blank">Phenol/Chloroform/Isoamyl alcohol</a></li>
<li>shaken vigorously inside 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 40 uL of phenol/chloroform/isoamyl alcohol</li>
<li>shaken vigorously inside fumehood for 15 seconds</li>
<li>centrifuged at 12000G at 4C for 2 min</li>
<li>aqueous layer was transferred to a new epp</li>
<li>100 uL of 96% ethanol was added and shaken</li>
<li>centrifuged at 21300G at 4C for 2min (this was a mistake and should have been 10 minutes)</li>
<li>The ethanol was removed and 100 ul of 70% ethanol solution was added to the sample.</li>
<li>centrifuged at 21300G at 4C for 5 min</li>
<li>The ethanol was removed and the palate was dried to air.</li>
<li>The pellet was resuspended in 50 uL milliQ. sample labeled: GFP, white box, freezer C1 in frank’s lab</li>
<li>Steps 1-13 were repeated with 30 uL FVIII mRNA taken from epps 2, 3, 4 (10 uL each)</li>
<li>The samples were analyzed on the regular agarose gel (see below)</li>
<p>Figure 1. Gel electrophoresis of the purified samples</p>
<p>
A small band is visible for the GFP, however, with a large smear above and below it. Factor 8 did not appear on the gel, however, as it was taken from an old epp, it may have been degraded prior to purification.
The likely explanation for the weak gel signal is the unsterile conditions of the pipette tips and epps as they have not been sterilized after previous use, or the old buffer used to run the gel.
</p>
<p><b>Conclusion:</b> More proper sterilization of the equipment is needed prior to every purification procedure. More mRNA of both GFP and factor 8 should be produced.</p>
