Difference between revisions of "Team:Bordeaux/Template:PolicyPracticesNotebook"

 
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             <h3> Notebook </h3>
 
             <h3> Notebook </h3>
  
 +
<H4><B> THE PDF VERSION OF THE NOTEBOOK IS IN PROGRESS. </B></H4>
 +
<H6><B>Thank you for your patience :)</B></H6>
  
−
<h5> Day 1 </h5>
 
  
−
<p> Description of everything we do  </p>
+
<ul>
 +
      <li><a href="https://static.igem.org/mediawiki/2015/e/e2/LAB_NOTEBOOK_JUNE_2015.pdf" target="_blank"><font size="3" ><B>LAB NOTEBOOK JUNE</B></font></a></li>
 +
      <li><a href="https://static.igem.org/mediawiki/2015/8/88/LAB_NOTEBOOK_JULY_2015.pdf" target="_blank"><font size="3" ><B>LAB NOTEBOOK JULY</B></font></a></li>
 +
      <li><a href="https://static.igem.org/mediawiki/2015/1/16/LAB_NOTEBOOK_AUGUST_2015_.pdf"target="_blank"><font size="3" ><B>LAB NOTEBOOK AUGUST</B></font></a></li>
  
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<TABLE BORDER="1">
+
  </ul>
−
  <CAPTION> Voici le titre du tableau </CAPTION>
+
−
  <TR>
+
−
<TH> Titre A1 </TH>
+
−
<TH> Titre A2 </TH>
+
−
<TH> Titre A3 </TH>
+
−
<TH> Titre A4 </TH>
+
−
  </TR>
+
−
  <TR>
+
−
<TH> Titre B1 </TH>
+
−
<TD> Valeur B2 </TD>
+
−
<TD> Valeur B3 </TD>
+
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<TD> Valeur B4 </TD>
+
−
  </TR>
+
−
</TABLE>  
+
  
−
<H5>02/06/2015</H5>
 
  
−
<H6>- Stock solution MgSO4  : 0,5M</H6>
+
</div>
−
<p>Pour 500mL :</p>
+
−
<p>61,62g de MgSO4 (MM : 246,48)</p>
+
−
<p>qsp 500mL H2O stérile</p>
+
  
−
<H6>- SOB Medium :  1 L</H6>
 
−
<TABLE BORDER="1">
 
−
  <TR>
 
−
<TH> Final [c] </TH>
 
−
<TH> Composent </TH>
 
−
<TH> Volume & Mass </TH>
 
−
  </TR>
 
−
  <TR>
 
−
<TH> 2% </TH>
 
−
<TD> Bactotryptone </TD>
 
−
<TD> 20 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TH> 10 mM </TH>
 
−
<TD> NaCl </TD>
 
−
<TD> 2 mL (5 M stock) </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TH> 2% </TH>
 
−
<TD> Yeast Extract </TD>
 
−
<TD> 5 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TH> 2,5 mM </TH>
 
−
<TD> KCl </TD>
 
−
<TD> 0,18 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TH> 10 mM </TH>
 
−
<TD> MgCl2 </TD>
 
−
<TD> 5 mL (2 M stock) </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TH> 10 mM </TH>
 
−
<TD> MgSO4 </TD>
 
−
<TD> 20 mL (0,5 M) </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TH> - </TH>
 
−
<TD> H2O </TD>
 
−
<TD> qsp 1L </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> pH media to 7 with NaOH, then autoclave </TD>
 
−
  </TR>
 
−
</TABLE>
 
  
 +
    </div>
 +
</section>
 +
</html>
  
−
<H6>- 2XTY Medium 500 mL</H6>
 
−
<TABLE BORDER="1">
 
−
  <TR>
 
−
<TH> Composent </TH>
 
−
<TH> Volume & Mass </TH>
 
−
  </TR>
 
−
  <TR>
 
−
<TD> Bactotryptone </TD>
 
−
<TD> 8 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> NaCl </TD>
 
−
<TD> 2,5 g</TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> Yeast Extract </TD>
 
−
<TD> 5 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> H2O </TD>
 
−
<TD>qsp 500 mL </TD>
 
−
  </TR>
 
−
</TABLE>
 
  
 +
<html>
  
−
<H5>03/06/2015</H5>
 
  
−
<H6>- Transformation Broth (TB):  1 L</H6>
 
  
−
<TABLE BORDER="1">
 
−
  <TR>
 
−
<TH> Final [c] </TH>
 
−
<TH> Composent </TH>
 
−
<TH> Volume & Mass </TH>
 
−
  </TR>
 
−
  <TR>
 
−
<TH> 10 mM </TH>
 
−
<TD> Pipes </TD>
 
−
<TD> 3,02 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TH> 15 mM </TH>
 
−
<TD> CaCl2 </TD>
 
−
<TD> 2,21 g  </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TH> 250 mM </TH>
 
−
<TD> KCl </TD>
 
−
<TD> 18,64 g </TD>
 
−
  </TR>
 
−
</TABLE>
 
−
<p>Mix the Pipes, CaCl2, and KCl in 900 ml of millipore water.  Add NaOH until pH is 6.7, </p>
 
−
<p>Don't worry, dust disappear after pH adjust.  </p>
 
−
<p>Add MnCl2 (see below), stir, adjust volume to 1 L, then filter sterilize. Store at 4C.</p>
 
−
     
 
−
<TABLE BORDER="1">
 
−
  <TR>
 
−
<TH> Final [c] </TH>
 
−
<TH> Composent </TH>
 
−
<TH> Volume & Mass </TH>
 
−
  </TR>
 
−
  <TR>
 
−
<TH> 55 mM </TH>
 
−
<TD> MnCl2 </TD>
 
−
<TD> 10,89 g </TD>
 
−
  </TR>
 
−
</TABLE>
 
  
  
−
<H6>- Making DMSO Competent Cells : DAY ONE</H6>
 
  
−
<p>Grow 12 ml overnight culture of favorite strain of E. coli in 2XTY at 37°C and 250 rpm (We use Denis’ DH5-alpha). </p>
+
<head>
 +
<!--head for calendar version of the notebook -->
 +
<!--
 +
<link rel='stylesheet' href='https://cdnjs.cloudflare.com/ajax/libs/fullcalendar/2.3.2/fullcalendar.css' />
 +
<script src='https://cdnjs.cloudflare.com/ajax/libs/jquery/2.1.4/jquery.min.js'></script>
 +
<script src='https://cdnjs.cloudflare.com/ajax/libs/moment.js/2.10.3/moment.min.js'></script>
 +
<script src='https://cdnjs.cloudflare.com/ajax/libs/fullcalendar/2.3.2/fullcalendar.js'></script>
 +
<script type='text/javascript' src='https://cdnjs/ajax/libs/fullcalendar/2.3.2/gcal.js'></script>
 +
-->
 +
<!--
 +
<script type='text/javascript'>
  
−
<H5>04/06/2015</H5>
+
$(document).ready(function() {
 +
  $('#calendar').fullCalendar({
 +
        events: [
 +
{
 +
title: 'Cell culture medium preparation',
 +
start: '2015-06-02',
 +
color: 'orange',
 +
textColor: 'black'
 +
},
 +
{
 +
title: 'Cell culture medium preparation',
 +
start: '2015-06-03',
 +
color: 'orange',
 +
textColor: 'black'
 +
},
 +
{
 +
title:'DMSO Competent cell',
 +
start:'2015-06-03',
 +
end:'2015-06-06',
 +
color: '#483D8B',
 +
textColor: 'White'
 +
        },
 +
{
 +
title: 'Cell culture medium preparation',
 +
start: '2015-06-08',
 +
color: 'orange',
 +
textColor: 'black'
 +
},
 +
{
 +
title: 'Making chemical competent cells',
 +
start: '2015-06-08',
 +
end:'2015-06-10',
 +
color: '#483D8B',
 +
textColor: 'White'
 +
},
 +
{
 +
title: 'Transformation Efficiency (Kit iGEM)',
 +
start: '2015-06-10',
 +
color: 'green',
 +
textColor: 'black'
 +
},
 +
{
 +
title: 'Transformation efficiency of Chemical competent cells',
 +
start: '2015-06-11',
 +
color: 'green',
 +
textColor: 'black'
 +
},
 +
{
 +
title: 'Glucose 2M stock preparation',
 +
start: '2015-06-11',
 +
color: '#F08080',
 +
textColor: 'black'
 +
},
 +
],
 +
  });
 +
  $('#calendar').fullCalendar('prev');
 +
});
 +
</script>
 +
-->
 +
<meta charset="utf-8">
 +
</head>
 +
<!--FIN HEAD FOR CALENDAR -->
  
−
<H6>- Making DMSO Competent Cells : DAY TWO</H6>
+
<!--
 +
//CALENDAR BODY
 +
<body>
 +
<div id='calendar'></div>
  
−
<p>Inoculate 1 L SOB with 12 ml overnight culture. </p>
+
</body>
−
<p>Keep 5mL of SOB for initial OD.</p>
+
//CALENDAR BODY END
−
<p>Grow culture at 18C and 180 rpm (this temperature is really important as we see a 10-fold decrease in competency when we grow them at room temperature).</p>
+
  
−
<H6>- Solution stock Chloramphenicol 34 mg.mL-1 (29,5 mL) :</H6>
+
-->
  
−
<p>Weigh out 1 g chloramphenicol and dissolve in 25 ml of 100% ethanol.</p>
 
−
<p>Make up volume to 29.5 ml with 100% ethanol.</p>
 
−
<p>Sterilize by filtration.</p>
 
−
 
−
<p>STORAGE : Aliquots of appropriate volume can be stored at -20 °C</p>
 
−
 
−
 
−
 
−
<H6>- Solution stock Tetracycline 12,5 mg.mL-1 (80 mL) :</H6>
 
−
 
−
 
−
<p>Weigh out 1 g tetracycline and dissolve in 75 ml of 1:1 vol/vol distilled water:ethanol.</p>
 
−
<p>Make up volume to 80 ml with 1:1 vol/vol distilled water:ethanol. (40mL ethanol + 40mL dH2O)</p>
 
−
<p>Sterilize by filtration.</p>
 
−
 
−
<p>STORAGE : Aliquots of appropriate volume should be wrapped in aluminium foil and stored at -20 °C</p>
 
−
 
−
<H5>05/06/2015</H5>
 
−
 
−
 
−
<H6>-LB agar 250mL (x3)</H6>
 
−
<TABLE BORDER="1">
 
−
  <TR>
 
−
<TH> Composent </TH>
 
−
<TH> Volume & Mass </TH>
 
−
  </TR>
 
−
  <TR>
 
−
<TD> Bactotryptone </TD>
 
−
<TD> 2,5 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> NaCl </TD>
 
−
<TD> 2,5 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> Yeast Extract </TD>
 
−
<TD> 1,25 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> Agar </TD>
 
−
<TD> 3 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> H2O </TD>
 
−
<TD> qsp 250 mL </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD>Adjust pH to 7.5 with NaOH and autoclave for 20 minutes </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> + Chloramphenicol (34 mg/ml in ethanol) </TD>
 
−
<TD> 75 µL / 250 mL medium {10 µg/ml (final)} </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> +Tetracycline (12,5 mg/ml in 50% ethanol)</TD>
 
−
<TD>250 µL / 250 mL medium {12,5 µg/ml (final)} </TD>
 
−
  </TR>
 
−
</TABLE>
 
−
 
−
<H6>-LB liquid 500mL </H6>
 
−
<TABLE BORDER="1">
 
−
  <TR>
 
−
<TH> Composent </TH>
 
−
<TH> Volume & Mass </TH>
 
−
  </TR>
 
−
  <TR>
 
−
<TD> Bactotryptone </TD>
 
−
<TD> 5 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> NaCl </TD>
 
−
<TD> 5 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> Yeast Extract </TD>
 
−
<TD> 2,5 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> H2O </TD>
 
−
<TD> qsp 500 mL </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD>Adjust pH to 7.5 with NaOH and autoclave for 20 minutes </TD>
 
−
  </TR>
 
−
<TABLE/>
 
−
 
−
 
−
<H6>- Making DMSO Competent Cells : DAY THREE</H6>
 
−
<p>Grow cells until A600 0.5-0.7</p>
 
−
<p>Blanck = SOB</p>
 
−
<p>A600 nm = 0,011</p>
 
−
<p>A600 nm = 0,008</p>
 
−
 
−
<p> => So we incubate all week-end at 18°C and 120 rpm.</p>
 
−
 
−
<H5>08/06/2015</H5>
 
−
 
−
<H6>- Making DMSO Competent Cells : DAY SIX</H6>
 
−
<p>Grow cells until A600 0.5-0.7</p>
 
−
<p>Blanck = SOB</p>
 
−
<p>A600 nm = 0,008</p>
 
−
 
−
<p>=> So we take an other protocol (openwarewet)</p>
 
−
 
−
<H6>-LB liquid 1L (x3) </H6>
 
−
<TABLE BORDER="1">
 
−
  <TR>
 
−
<TH> Composent </TH>
 
−
<TH> Volume & Mass </TH>
 
−
  </TR>
 
−
  <TR>
 
−
<TD> Bactotryptone </TD>
 
−
<TD> 10 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> NaCl </TD>
 
−
<TD> 10 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> Yeast Extract </TD>
 
−
<TD> 5 g </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD> H2O </TD>
 
−
<TD> qsp  1 L </TD>
 
−
  </TR>
 
−
<TR>
 
−
<TD>Adjust pH to 7.5 with NaOH and autoclave for 20 minutes </TD>
 
−
  </TR>
 
−
<TABLE/>
 
−
 
−
<H6>5X stock of M63 Medium (1L)</H6>
 
−
<p>- Add the following reagents to a 2-liter flask:</p>
 
−
<p>- 10 g (NH4)2SO4</p>
 
−
<p>- 68 g KH2PO4</p>
 
−
<p>- 2.5 mg FeSO4.7H2O</p>
 
−
<p>1 liter of high quality distilled water </p>
 
−
<p>Once the ingredients are added, heat with stirring until the components are completely dissolved. </p>
 
−
<p>Adjust to pH 7 with acid. </p>
 
−
<p>Autoclave at 121°C for 20 min. </p>
 
−
 
−
<p>PROBLEMS : we have FeSO4 precipitate + pH superior to 7 </p>
 
−
<p>Hypothesis : we had put 2,5g of FeSO4.7H2O instead of 2,5mg</p>
 
−
 
−
<H6>Preparation of other « Making Chemical Competent cells » DAY ONE</H6>
 
−
<p>TSS buffer</p>
 
−
<p>To make 50 mL:</p>
 
−
 
−
<p>5g PEG 8000 (Denis Friend)</p>
 
−
<p>1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H20)</p>
 
−
<p>Add LB to 50 mL</p>
 
−
<p>Filter sterilize (0.22 μm filter)</p>
 
−
<p>Add after sterilization 2.5 mL DMSO (the 09/06)</p>
 
−
 
−
<p>Overnight culture</p>
 
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<p>Grow a 5mL overnight culture of DH5-alpha in LB media at 37°C and 180 rpm.</p>
 
−
 
−
 
−
 
−
<H5>09/06/2015</H5>
 
−
<H6>Preparation of other « Making Chemical Competent cells » DAY TWO</H6>
 
−
 
−
<p>1. In the morning, dilute this culture back into 50mL of fresh LB media in a 200mL conical flask, at 37°C and 180 rpm. (Dilute the overnight culture by at least 1/100).</p>
 
−
 
−
<p>Grow the diluted culture to an OD600 of 0.2 - 0.5.</p>
 
−
<p>We obtained : OD600nm = 2,744</p>
 
−
<p>SO to obtain a good quantity of cells. We « dilute » culture later </p>
 
−
 
−
<p>2. Put eppendorf tubes on ice now. </p>
 
−
 
−
<p>- Theoretically, if your culture is 50 ml, you will need 50 tubes. But we had 5X more cells.  We will need 250 tubes.</p>
 
−
 
−
<p>- At this point you should also make sure that your TSS is being chilled (it stored at 4°C).</p>
 
−
 
−
<p>3. Split the culture into two 50mL falcon tubes and incubate on ice for 10 min. </p>
 
−
<p>TARE : tube 1 = 37,03g</p>
 
−
<p>        tube 2 = 37,00g</p>
 
−
 
−
<p>All subsequent steps should be carried out at 4°C and the cells should be kept on ice wherever possible</p>
 
−
 
−
<p>4. Centrifuge for 10 minutes at 3000 rpm and 4°C.</p>
 
−
 
−
<p>5. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.</p>
 
−
 
−
<p> 6. Resuspend in chilled TSS buffer. </p>
 
−
<p>The volume of TSS to use is 10% of the culture volume that you spun down => so 25 mL </p>
 
−
<p>You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall. We resuspend with </p><p>pipetteman with a cone cut at its end.</p>
 
−
 
−
<p>7. Add 100 μl aliquots to your chilled eppendorfs and store at − 80°C.</p>
 
−
<p>One part was stored directly at -80°C and the other part  was freeze in liquid nitrogen.</p>
 
−
 
−
<H5>10/06/2015</H5>
 
−
 
−
<H6>- Transformation Efficiency Kit iGEM</H6>
 
−
 
−
<H6>Protocol</H6>
 
−
 
−
 
−
<p> 1. Spin down the DNA tubes from the Transformation Efficiency Kit to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 rpm will be sufficient. Note: There should be 50 µL of DNA in each tube sent in the Kit.</p>
 
−
 
−
<p>2. Thaw competent cells on ice. Label one 2.0mL microcentrifuge tube for each concentration and then pre-chill by placing the tubes on ice.</p>
 
−
 
−
<p> 3. Pipet 1 µL of DNA into each microcentrifuge tube. For each concentration, use a separate tube.</p>
 
−
 
−
<p>4. Pipet 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. </p>
 
−
 
−
<p>5. Heat-shock the cells by placing into the waterbath for 1 minute. Be careful to keep the lids of the tubes above the water level, and keep the ice close by.</p>
 
−
 
−
<p> 6. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover.</p>
 
−
 
−
<p> 7. Add 200 µL of LB media per tube, and incubate at 37°C for 2 hours. </p>
 
−
 
−
<p>8. Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture.</p>
 
−
 
−
<p>9. Pipet 20 µL from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Do triplicates (3 each) of each tube if possible</p>
 
−
 
−
<p>10. Incubate at 37°C overnight. Position the plates so the agar side is facing up, and the lid is facing down.</p>
 
−
 
−
<p>11. Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency. If you've done triplicates of each sample, use the average cell colony count in the calculation.</p>
 
−
 
−
 
−
<IMG src='https://static.igem.org/mediawiki/2015/4/41/Capture_d%E2%80%99%C3%A9cran_2015-06-27_%C3%A0_16.07.10.png'>
 
−
</div>
 
−
    </div>
 
−
</section>
 
 
</html>
 
</html>

Latest revision as of 18:13, 13 September 2015

Notebook

THE PDF VERSION OF THE NOTEBOOK IS IN PROGRESS.

Thank you for your patience :)