Difference between revisions of "Team:Manchester/Project/Protocols"

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.agarinfo, .electrophoresisinfo, .glucoseinfo, .glycerolinfo, .mediuminfo, .SOBmediuminfo, .SOCmediuminfo, .TSSbufferinfo{
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.electrocompetentinfo, .agarinfo, .electrophoresisinfo, .glucoseinfo, .glycerolinfo, .mediuminfo, .SOBmediuminfo, .SOCmediuminfo, .TSSbufferinfo{
 
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  </div>
 
  </div>
 
</div>
 
</div>
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  <button class="electrocompetent sub_sub_title">Electrocompetent cells</button>
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<div class="electrocompetentinfo">
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  <p><i>Ingredients:</i></p>
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  <ul>
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    <li><i><a href="" target="_blank">LB media</a></i></li>
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    <li><i><a href="" target="_blank">50% (w/v) glycerol solution</a></i></li>
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  </ul>
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  <p><i>Before starting the procedure, be sure to:</i></p>
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  <ul>
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    <li><i><a href="" target="_blank">Pre-chill centrifuge and cool rotor to 4°C </a></i></li>
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    <li><i><a href="" target="_blank">Pre-chill 1L of 10% (w/v) glycerol solution on ice </a></i></li>
 +
    <li><i><a href="" target="_blank">Pre-chill 500mL/1000mL Falcon tubes on ice</a></i></li>
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  </ul>
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</div>
  
  
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             $(".TSSbufferinfo").toggle();
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        });
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});
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$(document).ready(function(){
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        $(".electrocompetent").click(function(){
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            $(".electrocompetentinfo").toggle();
 
         });
 
         });
 
});
 
});

Revision as of 15:14, 24 September 2016

Manchester iGEM 2016

Protocols banner

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Medium and Buffers


table 1
  1. Prepare the solution by mixing the ingredients stated above.
  2. Sterilize in an autoclave before using it to prepare the SOC medium.


table2
  1. Prepare the solution by mixing the ingredients stated above.
  2. Sterilize in an autoclave.

*Source from 2015 iGEM Exeter



For nucleic acids DNA/RNA separation

  • TAE buffer (Tris-acetate-EDTA)
  • TBE buffer (Tris-borate-EDTA)
  • LAB buffer (Lithium-acetate-borate)

table3
  1. Prepare the solution by mixing the ingredients stated above.


If not using pre-mixed LB agar powder, prepare the materials as below:


table4
  1. In a 1L Erlenmeyer flask, swirl and mix the solution.
  2. Cover the top of the flask with a lid/aluminum foil and label with autoclave tape.
  3. Autoclave the liquid setting for 20 minutes or according to your autoclave's specifications.
  4. After removing the solution from the autoclave, allow the agar solution to cool to 55°C in an oven or water bath.
  5. When pouring the LB agar into plates, keep the bench area sterile by working near a flame or Bunsen burner. Alternatively, prepare the plates in a vacuum hood.
  6. Add the appropriate amount of desired antibiotic (refer the table below) to the solution and swirl to mix.
  7. Pour approximately 20mL of LB agar per 10cm polystyrene Petri dish.
  8. Place the lids on the plates and allow them to cool for until the agar is solidified.
  9. Label the bottom of plates with antibiotic and date before storing in plastic bags or sealed with Parafilm at 4°C.
table 5

Table source from New England Biolabs


Additional note:

  • Antibiotic carbenicillin can be substituted for ampicillin in antibiotic selection plates [1].

Source from


If not using pre-mixed LB broth powder, prepare the materials as below:

table 6
  1. Prepare the solution by mixing the ingredients stated above.
  2. Sterilize in an autoclave.
table 7
  1. Prepare the solution by mixing the ingredients stated above.
  2. Sterilize in an autoclave before using it to prepare SOC medium.

Additional notes

  • Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20mM MgSO4.
  • SOB medium is also available dry pre-mixed from Difco, 0443-17
  • Adjust to pH 7.5 prior to use. This requires approximately 25mL of 1M NaOH per liter.

Source from OpenWetWare

table 8
  1. Prepare the solution by mixing the ingredients stated above.

*Source from OpenWetWare

table 9
  1. Prepare the solution by mixing the ingredients stated above.
  2. Filter sterilize using a 0.22 μm filter.
  3. Store at 4°C or -20°C.

*Source from OpenWetWare: TSS

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Transformation

Ingredients:

Before starting the procedure, be sure to:

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