http://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Notebook/Phage_Sensor/Saturday_7th_September.html&feed=atom&action=historyTeam:Paris Bettencourt/Notebook/Phage Sensor/Saturday 7th September.html - Revision history2024-03-29T13:55:10ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Notebook/Phage_Sensor/Saturday_7th_September.html&diff=171761&oldid=prevMarguerite: Created page with "<html> <div class ="tbnote"> <h2>Phage Sensor</h2> <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Phage_Sensor" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>..."2013-09-25T16:37:11Z<p>Created page with "<html> <div class ="tbnote"> <h2>Phage Sensor</h2> <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Phage_Sensor" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>..."</p>
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<h2>Phage Sensor</h2><br />
<a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Phage_Sensor" target="_blank" class="tbnotelogo PSlogo"> ASDF </a><br />
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<h3>Saturday 7<sup>th</sup> September</h3><br />
<p><b><em><br />
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Ligation, Making Electrocompetent Cells, Gibson Assembly, Transformation, Liquid culture of E1010<br />
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<b>Ligation</b><br><br />
<br><br />
<table border="1"><br />
<tr><br />
<td><b>Reagent</b></td><br />
<td><b>Volume</b></td><br />
</tr><br />
<tr><br />
<td>Vector</td><br />
<td>1 µl</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4.5 µl</td><br />
</tr><br />
<tr><br />
<td>Fermentas T4 Ligase Buffer</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Fermentas T4 Ligase Enzyme</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td><b>Total Volume</b></td><br />
<td>10 µl</td><br />
</tr><br />
</table><br><br />
<br><br />
SPCR5, pSB1C3<br><br />
SPCR11, pSB1A3, pSB1C3<br><br />
SPCR13, pSB1A3, pSB1C3<br><br />
SPCR2, pSB1A3, pSB1C3<br><br />
<br><br />
Incubate at room temperature for 10 minutes.<br><br />
<br><br />
<br><br />
<b>Making Electrocompetent Cells</b><br><br />
1) Start a liquid culture of cells.<br><br />
NEB turbo<br><br />
2) dilute the cells 100X.<br><br />
100 ml LB.<br><br />
Grow at 30°C for about 90 minutes. <br><br />
3) Harvest the cells.<br><br />
When the cells reach an OD600 of between 0.6 and 0.8<br><br />
Split the culture into 2x 50 ml falcon tubes, on ice.<br><br />
Centrifuge at 4 °C for 10 min at 4000 rpm.<br><br />
5) Wash and combine the cells.<br><br />
Remove the supernatant.<br><br />
Resuspend the cells in 2x 25 ml of ice cold water.<br><br />
Combine the volumes in a single 50 ml falcon tube.<br><br />
6) Wash the cells 2 more times.<br><br />
Centrifuge at 4 °C for 10 min at 4000 rpm.<br><br />
Resuspend in 50 ml of ice cold water.<br><br />
Repeat.<br><br />
7) Wash and concentrate the cells for electroporation.<br><br />
Centrifuge at 4 °C for 10 min at 4000 rpm.<br><br />
Resuspend in 1-2 ml of ice cold water.<br><br />
We will use 200 ul of washed cells per transformation.<br><br />
<br><br />
<br><br />
<br><br />
<b>Gibson Assembly</b><br><br />
<br><br />
Amount per Reaction<br><br />
Positive Control**<br><br />
<br><br />
<br><br />
PCR Fragment(s)<br><br />
+ linearized vector<br><br />
2-10 μl (0.02–0.5 pmols)*<br><br />
<br><br />
<br><br />
Gibson<br><br />
Assembly Master<br><br />
Mix (2X)<br><br />
10 μl<br><br />
<br><br />
<br><br />
Deionized H2O<br><br />
XX μl<br />
<br><br />
<br><br />
Total Volume<br><br />
20 μl<br><br />
<br><br />
<br><br />
<br><br />
Incubate samples in a thermocycler at 50°C for 15 minutes. <br><br />
Put on Ice<br><br />
<br><br />
<br><br />
<b>Transformation</b><br />
pS002 (Chl), pS002 (Amp), pS003 (Amp), pS004 (Chl), pS004 (Amp), pS005 (Chl), pS006-7 (Chl), pS007 (Chl), pS008 (Amp), pS009 (Chl), pS012 (Amp), pS013 (Chl), pS013 (Amp) <br><br />
<br><br />
1) Prepare BD tubes with a pipette filled with LB at the interior of each tube (pipette supplied with the electroporation cuvette)<br><br />
<br><br />
2) Test the purity of the electrocompetent cells.<br><br />
Add 200 ul of washed cells to a cuvette.<br><br />
<br><br />
3) Mix the cells and DNA in a cuvette.<br><br />
200 ul of washed cells with 200 ng of PCR product.<br><br />
Keep the cuvette on ice until just before the electroporation.<br><br />
<br><br />
4) Preload a pipette with 1 ml of LB.<br><br />
<br><br />
5) Pulse the cuvette with voltage.<br><br />
Dry the electrodes with a kimwipe prior to loading.<br><br />
Use the EC2 setting.<br><br />
<br><br />
6) Listen for arcing.<br><br />
A cracking sound means all the cells are dead.<br><br />
Note the time constant: 5 is good, 5.8 is great.<br><br />
<br><br />
7) Immediately recover the cells.<br><br />
Add the 1 ml of preloaded LB and pipet up and down to mix.<br><br />
Collect 1 ml of cells, some volume is lost in the cuvette.<br><br />
<br><br />
8) Incubate 1/2 h at 37 °C with shaking.<br><br />
<br><br />
9) Plate 10/200 ul of recovered cells on selective plates.<br><br />
Use antibiotic appropriate to the part being integrated.<br><br />
Let the other 900 ul rest overnight at room temperature.<br><br />
<br><br />
10) Concentrate and plate the remaining cells<br><br />
Spin down quickly and resuspend in 100 ul LB before plating.<br><br />
<br><br />
Transformed cells should be incubated at 37 °C.<br><br />
Colonies should appear 24-48 h after plating.<br><br />
<br><br />
<b>Liquid culture of E1010</b><br><br />
<br />
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</html></div>Marguerite