From d67dfbb4fea61151d4503eeace74e0be9e287258 Mon Sep 17 00:00:00 2001
From: Oliver Richter <oliverrichter224@gmail.com>
Date: Sun, 1 Dec 2024 15:59:45 +0000
Subject: [PATCH] Update file engineering.html
---
wiki/pages/engineering.html | 99 +++++++++++++++++++++++++++++++++----
1 file changed, 89 insertions(+), 10 deletions(-)
diff --git a/wiki/pages/engineering.html b/wiki/pages/engineering.html
index 9f2b173..ef6f27a 100644
--- a/wiki/pages/engineering.html
+++ b/wiki/pages/engineering.html
@@ -351,17 +351,96 @@ Despite working carefully and strictly following the protocol instructions, our
<h4>Build</h4>
<p>
We, therefore, thought about how we could optimize the Quiagen MinElute Gel Extraction Kit ourselves and came up with the following new approaches:
- <br>
- - additional washing step with 70 % ethanol <br>
-- let the alcoholic components evaporate by letting the tubes stand for 15-20 min with an open lid <br>
-- after the evaporation transfer the spin column in a new tube <br>
-- elute with 20 µL Elution Buffer <br>
-- run PCR as usual <br>
-- Before loading the PCR samples, let the samples again stand with an open lid for about 10-15 min <br>
-- add the loading dye pipette up and down and let it stand again for 5 min <br>
-- Pipette up and down before loading the samples until loading dye and sample are well mixed <br>
+ </p>
+ <ul>
+ <li>additional washing step with 70 % ethanol</li>
+ <li>let the alcoholic components evaporate by letting the tubes stand for 15-20 min with an open lid</li>
+ <li>after the evaporation transfer the spin column in a new tube</li>
+ <li>elute with 20 µL Elution Buffer</li>
+ <li>run PCR as usual</li>
+ <li>Before loading the PCR samples, let the samples again stand with an open lid for about 10-15 min</li>
+ <li>add the loading dye pipette up and down and let it stand again for 5 min</li>
+ <li>Pipette up and down before loading the samples until loading dye and sample are well mixed</li>
+ </ul>
+ <p>
+ The working group of Prof. Landau, also located at the CSSB, kindly provided us with 70 % ethanol, enabling us to test our new approaches to improving the gel clean-up. We had enough of the other necessary materials, such as tubes, which accelerated the implementation of our ideas.
+ </p>
-The working group of Prof. Landau, also located at the CSSB, kindly provided us with 70 % ethanol, enabling us to test our new approaches to improving the gel clean-up. We had enough of the other necessary materials, such as tubes, which accelerated the implementation of our ideas.
+ <h4>Test</h4>
+ <p>
+ As we received the 70 % ethanol we tested our newly designed protocol. The instruction of Quiagen are marked in black and our additional steps are marked in green.
+ </p>
+ <ol>
+ <li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.</li>
+ <li>Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg gel ~ 100 µL). The maximum amount of gel slice per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.</li>
+ <li>Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min during incubation to help dissolve the gel.</li>
+ <li>Alter the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 pl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.</li>
+ <li>Add 1 gel volume of isopropanol to the sample and mix by inverting</li>
+ <li>Place a MinElute spin column • in a provided 2 mL collection tube or A into a vacuum manifold. For information about set up, see the MinElute Handbook.</li>
+ <li>Apply sample to the MinElute column and • centrifuge for 1 min or A apply vacuum until the entire sample has passed through the column. • Discard flow-through and place the MinElute column back into the same collection tube. For sample volumes of more than 800 µL, simply load and spin again.</li>
+ <li>Add 500 µL Buffer QG to the MinElute column and • centrifuge for 1 min or A apply vacuum. • Discard flow-through and place the MinElute column back into the same collection tube.</li>
+ <li>Add 750 µL Buffer PE to MinElute column and centrifuge for 1 min or A apply vacuum. • Discard flow-through and place the MinElute column back into the same collection tube. Note: If the DNA will be used for salt-sensitive applications, such as direct sequencing and blunt-ended ligation</li>
+ <li>Add 750 µL 70% ethanol to MinElute column and centrifuge for 1 min</li>
+ <li>Let the alcoholic components evaporate by letting the tubes stand for 15-20 min with an open lid</li>
+ <li>Transfer the MinElute column after the evaporation in a new 2 mL tube</li>
+ <li>Centrifuge the column in a 2 mL collection tube (provided) for 1 min. Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.</li>
+ <li>Place each MinElute column into a clean 1.5 ml microcentrifuge tube. To elute DNA, add 20 µL instead of 10 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of the MinElute membrane. (Ensure that the elution buffer is dispensed directly onto the membrane for complete elution of bound DNA.) Let the column stand for 1 min, and then centrifuge the column for 1 min.</li>
+ <li>Measure concentration and 260/280 and 260/230 ratios using a NanoDrop</li>
+ <li>run PCR as normal</li>
+ <li>Let the samples stand with an open lid for about 10-15 min before loading the PCR samples</li>
+ <li>Add the loading dye and pipette up and down to suspend </li>
+ <li>Let it stand again with an open lid for 5 min</li>
+ <li>Pipette up and down before loading the samples until loading dye and sample are well mixed</li>
+ </ol>
+ <p>
+ First, we tested the optimized clean-up protocol using pUC_HasA and pMK1 samples, which we amplified by PCR and then loaded onto a 1% agarose gel. We can show that we increased the purity regarding contamination with salts and organic solvent.
+ </p>
+ <table border="1" cellpadding="5" style="border-collapse: collapse; width: 100%;">
+ <tbody>
+ <tr style="font-weight: bold;">
+ <td>additional washing step with 70% ethanol</td>
+ <td>Sample</td>
+ <td>Concentration ng/µL</td>
+ <td>260/280</td>
+ <td>260/230</td>
+ </tr>
+ <tr>
+ <td>yes</td>
+ <td>pUC_HasA 1</td>
+ <td>53.6</td>
+ <td>1.77</td>
+ <td>1.25</td>
+ </tr>
+ <tr>
+ <td>no</td>
+ <td>pUC_HasA 2</td>
+ <td>64.8</td>
+ <td>1.72</td>
+ <td>0.13</td>
+ </tr>
+ <tr>
+ <td>yes</td>
+ <td>pMK1 1</td>
+ <td>16.4</td>
+ <td>1.47</td>
+ <td>0.75</td>
+ </tr>
+ <tr>
+ <td>no</td>
+ <td>pMK1 2</td>
+ <td>30.9</td>
+ <td>1.42</td>
+ <td>0.15</td>
+ </tr>
+ </tbody>
+ </table>
+ <p>
+ After following our optimized protocol, we were first able to load our beforehand extracted and then amplified resilin DNA sequence on an agarose gel (Fig. 25) and then move on to the next steps in cloning resilin, which was extracting the amplified resilin sequence and starting a restriction-ligation reaction with pET28c(+), followed by transformation into <i>E. coli</i>.
+ </p>
+
+ <h4>Engineering Success</h4>
+ <p>
+ We learned that the elongated, additional evaporation steps and the 5-min incubation with the loading dye before loading the samples on the agarose gel were significant in avoiding the samples floating out of the gel pockets. We are really proud of our engineering success because it allows us to move on with our project and especially bring the cloning of our resilin repeats forward.
</p>
</div>
--
GitLab