There are many ways to prepare competent cells. One such way is through the use of CaCl2, which keeps the cell wall more permeable, facilitating the entrance of circular DNA.
There are many ways to prepare competent cells. One such way is through the use of CaCl<sub>2</sub>, which keeps the cell wall more permeable, facilitating the entrance of circular DNA.
</p>
<ol>
<li>Make an overnight culture of <em>E. coli</em> cells, keeping it at 37ºC, with shaking.</li>
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@@ -175,10 +175,10 @@
<li>Incubate cells on ice for 15min.</li>
<li>Centrifuge at 3500rpm, at 4ºC for 5min.</li>
<li>Discard the supernatant.</li>
<li>Resuspend pellet with a micropipette, in 100microliters of 0.1M CaCl2, kept at 4ºC before use.</li>
<li>Resuspend pellet with a micropipette, in 100microliters of 0.1M CaCl<sub>2</sub>, kept at 4ºC before use.</li>
<li>Incubate the cells on ice for 30min.</li>
<li>Centrifuge at 3500rpm, at 4ºC for 5min.</li>
<li>Resuspend the cells in 2 mL ice-cold 0.1 M CaCl2/20% glycerol.</li>
<li>Resuspend the cells in 2 mL ice-cold 0.1 M CaCl<sub>2</sub>/20% glycerol.</li>
<li>Incubate the cells for 45min on ice.</li>
</ol>
<p>
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@@ -210,7 +210,7 @@
<li>To two separate tubes, add 500 ng of DNA of each sample (one sample being plasmid containing target sequence, the other sample being the backbone said target sequence will later be ligated into).</li>
<li>Add 5 μl of 10x reaction buffer compatible with both enzymes, to both DNA mixtures separately.</li>
<li>Add 1 μl of each of the endonucleases to each mixture.</li>
<li>Add ddH2O to a final volume of 50 μl. </li>
<li>Add ddH<sub>2</sub>O to a final volume of 50 μl. </li>
<li>Tap on the tubes to mix. Centrifuge for a few seconds to spin down liquid.</li>
<li>Incubate at 37°C for 30 min. </li>
<li>Heat-inactivate the enzymes at 80°C for 20 min.</li>
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@@ -238,7 +238,7 @@
<li>Mix 20 ng of each digestion mixture.</li>
<li>Add 2 μl of 10x reaction for T4 DNA ligase.</li>
<li>Add 1 μl of T4 DNA ligase.</li>
<li>Add ddH2O to a final volume of 20 μl.</li>
<li>Add ddH<sub>2</sub>O to a final volume of 20 μl.</li>
<li>Incubate at room temperature (~22°C) for 30 min. </li>
<li>Heat-inactivate the enzymes at 80°C for 20 min.</li>
</ol>
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<h4>Day 2:</h4>
<ol>
<li>Inoculate the O.N culture 1:100 into LB/antibiotic media.</li>
<li>Shake at 37℃, until desired OD600, add desired concentration of inducer directly into the culture (same volume of dH2O in uninduced controls), put in desired temperature, 220rpm shaking, start induction for desired time.</li>
<li>Shake at 37℃, until desired OD600, add desired concentration of inducer directly into the culture (same volume of dH<sub>2</sub>O in uninduced controls), put in desired temperature, 220rpm shaking, start induction for desired time.</li>
<li>After induction, harvest and lyse the culture by either sonication or SDS (not for purification).</li>
<h5>For Purification also do:</h5>
<li>After sonication, centrifugate cell lysates at 14000 rpm for 10 min at 4 °C, collect supernatants and filter through 0.2 μm filters and keep on ice.</li>
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<li>Wash the column with Buffer A 20mL (or 5x volume) </li>
<li>Elute with Buffer A containing 20mM, 50mM, 100mM, 250mM, 500mM imidazole. 16mL each concentration. Collect the flow through by Eppendorf tube, 1mL/tube (normally the protein will be eluted at 250mM and 500mM, but collect all concentrations at first time), put on ice.</li>
<li>Wash the column with 5x volume Buffer A containing 500mM imidazole.</li>
<li>Wash the column with 5x volume of ddH2O.</li>
<li>Wash the column with 5x volume of ddH<sub>2</sub>O.</li>
<li>Wash the column with 5x volume Buffer A, half fill the column, keep at 4℃.</li>
</ol>
</li>
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@@ -463,7 +463,7 @@
</table>
</div>
<li>Mix gently and spin down briefly.</li>
<li>Incubate at the optimal reaction temperature (often 37 deg. celsius) for 1-16 hours.</li>
<li>Incubate at the optimal reaction temperature (often 37 ℃) for 1-16 hours.</li>
</ol>
</div>
</div>
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@@ -507,10 +507,10 @@
<div>
<p>(Protocol modified from GeneArt™ Gibson Assembly® HiFi Master Mix from Thermo Fisher Scientific)</p>
<ol>
<li>Assembly mix: Mix 0.08 pmol of each DNA insert and the vector. Double acquired volume with the provided 2X GeneArt™ Gibson Assembly® HF Master Mix. Add dH2O up to 20 uL.</li>
<li>Assembly mix: Mix 0.08 pmol of each DNA insert and the vector. Double acquired volume with the provided 2X GeneArt™ Gibson Assembly® HF Master Mix. Add dH<sub>2</sub>O up to 20 uL.</li>
<li>Positive control: Mix 10 uL of the provided positive control with 10 uL 2X GeneArt™ Gibson Assembly® HF Master Mix.</li>
<li>Gently vortex the samples and spin down at 4000 rpm for 15s.</li>
<li>Incubate at 50 oC for 15 min</li>
<li>Incubate at 50 <sup>o</sup>C for 15 min</li>
<li>Screening: Transform cells and plates with proper antibiotics (positive control on kanamycin and ampicillin).</li>
</ol>
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@@ -537,10 +537,10 @@
<li>
Make 3 mL O/N cultures (with appropriate antibiotic for the current bacteria). Amounts found below:<br>
<olstyle="list-style-type: lower-latin;">
<li>12 ug/mL tetracycline</li>
<li>100 ug/mL ampicillin</li>
<li>25 ug/mL chloramphenicol</li>
<li>50 ug/mL kanamycin</li>
<li>12 µg/mL tetracycline</li>
<li>100 µg/mL ampicillin</li>
<li>25 µg/mL chloramphenicol</li>
<li>50 µg/mL kanamycin</li>
</ol>
</li>
<li>Extract plasmids with “GeneJET PCR Purification Kit” from Thermo Scientific (see protocol 11 for link to download)</li>
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@@ -586,8 +586,8 @@
<p><b>Quick version</b></p>
<ol>
<li>Staining solution was added to the gel and microwaved for 30 s and then incubated on a shaker for 20 min</li>
<li>The gel was washed with dH2O and ~30 mL destaining solution was added. Microwaved for 45 s and then incubated on a shaker for ~20 min. This step was repeated until a desired destained was achieved.</li>
<li>The gel was washed with dH2O again</li>
<li>The gel was washed with dH<sub>2</sub>O and ~30 mL destaining solution was added. Microwaved for 45 s and then incubated on a shaker for ~20 min. This step was repeated until a desired destained was achieved.</li>
<li>The gel was washed with dH<sub>2</sub>O again</li>
</ol>
</div>
</div>
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</table>
</div>
<p>All PCR reactions were performed in 50 μl, containing 25 μl of Q5 high fidelity 2x Master Mix from NEB, 0.25 μl of forward primer, 0.25 μl of reverse primer, 1 μl of template DNA (1 ng/μl) and 23.5 μl of ddH2O.</p>
<p>All PCR reactions were performed in 50 μl, containing 25 μl of Q5 high fidelity 2x Master Mix from NEB, 0.25 μl of forward primer, 0.25 μl of reverse primer, 1 μl of template DNA (1 ng/μl) and 23.5 μl of ddH<sub>2</sub>O.</p>
<h3>PCR reaction mixture for touchdown, gradient and limited gradient:</h3>
<divclass="centerAll">
<table>
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@@ -728,7 +728,7 @@
</div>
<h3>Colony PCR programme:</h3>
<p>Each tube for PCR reaction contained 4 μl of 5x Phusion HF buffer (Thermo Scientific), 0.4 μl of 10 mM dNTPs, 0.5 μl of forward primer, 0.5 μl of reverse primer, 14.4 μl of ddH2O, 0.2 μl of Phusion High-Fidelity DNA Polymerase (Thermo Scientific) and a scraped piece of desired colony as template. Total volume was 20 μl.</p>
<p>Each tube for PCR reaction contained 4 μl of 5x Phusion HF buffer (Thermo Scientific), 0.4 μl of 10 mM dNTPs, 0.5 μl of forward primer, 0.5 μl of reverse primer, 14.4 μl of ddH<sub>2</sub>O, 0.2 μl of Phusion High-Fidelity DNA Polymerase (Thermo Scientific) and a scraped piece of desired colony as template. Total volume was 20 μl.</p>
<p>For the enzymatic assay of PETase activity two different types of pretreatment of PET were used, DMSO and triton treatment.</p>
<h3>DMSO treatment:</h3>
<br><br>
<p>Samples with DMSO contained 12 ml of cell lysate and 3 ml of DMSO Mix (42 % (v/v) DMSO, 250 mM glycine-NaOH (pH 9), 250 mM NaCl).</p>
<p>Triton treatment was derived from the TJUSLS China 2021 iGEM team. PET used in samples treated with triton was incubated for 30 minutes in 50 °C in a mixture containing 0.5% (v/v) Triton X-100 and 10 mM Na2CO3. PET was then air-dried in 37 °C and put in samples containing 12 ml of cell lysates and 3 ml of glycine NaOH (pH 9).</p>
<p>Samples were incubated at different conditions and with different types of PET (see results) and samples with uninduced PETase cells and with induced cells expressing IF3 protein were incubated in all the stated conditions and used as negative controls.Samples for measurement were taken every few hours in the course of 3 days and every time sample was taken, heat inactivated at 85 °C for 10 mins and centrifuged (4000 rpm, 5 mins) to dispose of cell debris and PET powder and measured spectrophotometrically.</p>
