Difference between revisions of "Team:Ionis Paris/Protocol 11"

Line 51: Line 51:
 
                               <h4 class="blog" style="margin-top:20px">
 
                               <h4 class="blog" style="margin-top:20px">
 
                                     Glycerol Freezing Solution (50%)</h4>
 
                                     Glycerol Freezing Solution (50%)</h4>
−
                                     <p>Prepare a 50% glycerol stock solution by mixing 5ml glycerol with 5ml distilled, sterile water.<br/>
+
                                     <p>Prepare a 50% glycerol stock solution by mixing 5mL glycerol with 5mL distilled, sterile water.<br/>
 
Mix well to make sure that you see one uniform solution, and there are no layers present. <br/>
 
Mix well to make sure that you see one uniform solution, and there are no layers present. <br/>
 
</p>
 
</p>
Line 57: Line 57:
  
 
                                 <h4 class="blog" style="margin-top:20px">Bacteria storage</h4>
 
                                 <h4 class="blog" style="margin-top:20px">Bacteria storage</h4>
−
                                     <p>Mix 50 µL of the overnight culture of bacteria of interest with 50 µL 50% glycerol solution (25% glycerol final concentration) into a 2 mL cryotube<br/>
+
                                     <p>Mix 50µL of the overnight culture of bacteria of interest with 50µL 50% glycerol solution (25% glycerol final concentration) into a 2mL cryotube<br/>
−
<b>NB : Snap top tubes are not recommended as they can open unexpectedly at -80°C</b><br/>
+
<i>NB : Snap top tubes are not recommended as they can open unexpectedly at -80°C </i> <br/>
−
Label both the lid and the tube of the cryotube. Frozen tubes are hard to write on and samples stored for long periods at -80°C can lose labels stuck to tube.
+
Label both the lid and the tube of the cryotube. Frozen tubes are hard to write on and samples stored for long periods at -80°C can lose labels stuck to tube.  
 
Store at -80°C
 
Store at -80°C
 
</p>
 
</p>
 
                                 <h4 class="blog" style="margin-top:20px">Bacteria recovery</h4>
 
                                 <h4 class="blog" style="margin-top:20px">Bacteria recovery</h4>
−
                                 <p>Open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw.<br/>
+
                                 <p>Pipet bacteria cells and put it into a 500mL sterile Erlenmeyer containing 100mL of sterile liquid LB medium. Cover with aluminum paper and place the Erlenmeyer in a rotative incubator at 37 Celsius degrees, 120rpm O/N.</p>
−
Either streak the bacteria onto an LB agar plate (see culture medium preparation protocol) and grow your bacteria overnight at the appropriate culture condition. The next day you will be able to start an overnight culture for plasmid DNA prep the following day.<br/>
+
−
Or scrub a bit of bacteria cell and put it into a 500mL sterile Erlenmeyer containing 100mL of sterile liquid LB medium. Cover with aluminum paper and place the Erlenmeyer in a rotative incubator at 37 Celsius degrees, 120rpm.</p>
+
  
  
Line 237: Line 235:
 
               <p><li><a href="https://www.addgene.org/plasmid-protocols/create-glycerol-stock/
 
               <p><li><a href="https://www.addgene.org/plasmid-protocols/create-glycerol-stock/
 
">Addgene protocol for creating a Glycerol stock</a></li><br>
 
">Addgene protocol for creating a Glycerol stock</a></li><br>
−
                   <li><a href="http://www.sigmaaldrich.com/technical-documents/protocols/biology/restriction-enzyme-cloning-manual-transformation.html#sthash.FmjdaJmZ.dpuf">Sigma-Aldrich protocol for E.coli manipulations
+
                   <li><a href="http://www.sigmaaldrich.com/technical-documents/protocols/biology/restriction-enzyme-cloning-manual-transformation.html#sthash.FmjdaJmZ.dpuf">Sigma-Aldrich protocol for E.coli manipulations </a> </li></p>
−
</a> </li><br>
+
 
−
                  <li><a href="http://www.addgene.org/plasmid-protocols/dna-ligation/">Addgene protocol for DNA ligation</a> </li><br>
+
−
                <li><a href="http://www.scielo.cl/scielo.php?pid=s0717-34582005000100014&script=sci_arttext"> 
+
−
Tu, Z. et al. An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains. Electronic Journal of Biotechnology 8, (2005).
+
−
</a> </li></p>
+
 
                         </aside>
 
                         </aside>
 
                     </div>
 
                     </div>

Revision as of 19:22, 16 October 2016

Protocol 11 : Glycerol storage

Aim: Safely store cells at -80°C

Following steps will be performed under a class II PSM

Glycerol Freezing Solution (50%)

Prepare a 50% glycerol stock solution by mixing 5mL glycerol with 5mL distilled, sterile water.
Mix well to make sure that you see one uniform solution, and there are no layers present.

Bacteria storage

Mix 50µL of the overnight culture of bacteria of interest with 50µL 50% glycerol solution (25% glycerol final concentration) into a 2mL cryotube
NB : Snap top tubes are not recommended as they can open unexpectedly at -80°C
Label both the lid and the tube of the cryotube. Frozen tubes are hard to write on and samples stored for long periods at -80°C can lose labels stuck to tube. Store at -80°C

Bacteria recovery

Pipet bacteria cells and put it into a 500mL sterile Erlenmeyer containing 100mL of sterile liquid LB medium. Cover with aluminum paper and place the Erlenmeyer in a rotative incubator at 37 Celsius degrees, 120rpm O/N.