Difference between revisions of "Team:Calgary/Protocols"

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                                         <li>Pipette up and down 3-5 times.</li>
 
                                         <li>Pipette up and down 3-5 times.</li>
 
                                         <li>Incubate at room temperature for 10 minutes.</li>
 
                                         <li>Incubate at room temperature for 10 minutes.</li>
−
                                         <li>Transform cells with 1μL of rehydrated DNA as per transformation protocol. Store the remaining amount at
+
                                         <li>Transform cells with 1μL of rehydrated DNA as per transformation protocol. Store the remaining amount at -20°C.</li>
−
                                            -20°C.</li>
+
 
                                     </ol>
 
                                     </ol>
 
                                 </td>
 
                                 </td>
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                                               <li>15% (w/v) agar</li>
 
                                               <li>15% (w/v) agar</li>
 
                                   </ul>
 
                                   </ul>
−
                                     <p>Appropriate antibiotic:</p>                                  
+
                                     <p>Appropriate antibiotic:</p>  
 +
                                  <ul>
 +
                                              <li>Ampicillin (final concentration of 100μg/mL)</li>
 +
                                              <li>Chloramphenicol (final concentration of 25μg/mL)</li>
 +
                                              <li>Kanamycin (final concentration of 50μg/mL)</li>                           
 +
                                  </ul>
 +
                                  <p>dH₂O</p>
 +
                                  <p>1500mL Erlenmeyer flask</p>
 +
                                  <p>Stir bar</p>
 +
                                  <p>Aluminum foil</p>
 
                                 </td>
 
                                 </td>
 
                             </tr>
 
                             </tr>
 
                             <tr>
 
                             <tr>
 
                                 <td>
 
                                 <td>
−
                                     <h5>Protocols</h5>
+
                                     <h5>Protocol</h5>
 
                                 </td>
 
                                 </td>
 
                                 <td>
 
                                 <td>
 
                                     <ol>
 
                                     <ol>
−
                                         <li>Add 10μL of ddH₂O to the desired well.</li>
+
                                         <li>In a 1500mL Erlenmeyer flask, add 10g tryptone, 5 g yeast extract, 10g NaCl and 15g agar. Dissolve solids in 1000mL dH₂O and add a stir bar</li>
−
                                         <li>Pipette up and down 3-5 times.</li>
+
                                         <li>Cover flask loosely with aluminum foil, secure with autoclave tape and sterilize by autoclaving</li>
−
                                         <li>Incubate at room temperature for 10 minutes.</li>
+
                                         <li>Remove agar from autoclave and allow agar to cool until warm to the touch before adding appropriate antibiotic</li>
−
                                         <li>Transform cells with 1μL of rehydrated DNA. Store the remaining amount at
+
                                         <li>Stir on hot plate and magnetic stirrer for 30 seconds</li>
−
                                            -20°C.</li>
+
                                      <li>Pour agar into plates using aseptic technique</li>
 
                                     </ol>
 
                                     </ol>
 
                                 </td>
 
                                 </td>

Revision as of 23:30, 15 October 2018

Team:Calgary/Notebook - 2018.igem.org

PROTOCOLS



Below are the protocols used by the team.

Materials

iGEM 2018 distribution kit

  • List Item 1
  • List Item 2

ddH₂O
Protocol
  1. Add 10μL of ddH₂O to the desired well.
  2. Pipette up and down 3-5 times.
  3. Incubate at room temperature for 10 minutes.
  4. Transform cells with 1μL of rehydrated DNA as per transformation protocol. Store the remaining amount at -20°C.

Materials

Synthesized DNA from IDT or Genscript

ddH₂O
Protocol
  1. Centrifuge tube containing the synthesized DNA for 1 minute at 3000g.
  2. Add 20μL ddH₂O.
  3. Vortex for 1 minute.
  4. Incubate at 50°C for 15 minutes.
  5. Briefly centrifuge. Store at -20°C.

Materials

Luria-Bertani broth with agar:

  • 10% (w/v) tryptone
  • 5% (w/v) NaCl
  • 10% (w/v) yeast extract
  • 15% (w/v) agar

Appropriate antibiotic:

  • Ampicillin (final concentration of 100μg/mL)
  • Chloramphenicol (final concentration of 25μg/mL)
  • Kanamycin (final concentration of 50μg/mL)

dH₂O

1500mL Erlenmeyer flask

Stir bar

Aluminum foil
Protocol
  1. In a 1500mL Erlenmeyer flask, add 10g tryptone, 5 g yeast extract, 10g NaCl and 15g agar. Dissolve solids in 1000mL dH₂O and add a stir bar
  2. Cover flask loosely with aluminum foil, secure with autoclave tape and sterilize by autoclaving
  3. Remove agar from autoclave and allow agar to cool until warm to the touch before adding appropriate antibiotic
  4. Stir on hot plate and magnetic stirrer for 30 seconds
  5. Pour agar into plates using aseptic technique

Experimental Details

Registry DNA was rehydrated for completion of the Interlab Study. Also, Part:BBa_K934001 (phaC1-A-B1) was rehydrated and transformed into our chassis so that PHB was produced and preliminary secretion assays could be performed before the Synthesis subgroup had completed their cloning.
Materials
  • iGEM 2017 distribution kit
  • ddH₂O
Protocols
  1. Add 10μL of ddH₂O to the desired well.
  2. Pipette up and down 3-5 times.
  3. Incubate at room temperature for 10 minutes.
  4. Transform cells with 1μL of rehydrated DNA. Store the remaining amount at -20°C.

Experimental Details

Registry DNA was rehydrated for completion of the Interlab Study. Also, Part:BBa_K934001 (phaC1-A-B1) was rehydrated and transformed into our chassis so that PHB was produced and preliminary secretion assays could be performed before the Synthesis subgroup had completed their cloning.
Materials
  • iGEM 2017 distribution kit
  • ddH₂O
Protocols
  1. Add 10μL of ddH₂O to the desired well.
  2. Pipette up and down 3-5 times.
  3. Incubate at room temperature for 10 minutes.
  4. Transform cells with 1μL of rehydrated DNA. Store the remaining amount at -20°C.

Experimental Details

Registry DNA was rehydrated for completion of the Interlab Study. Also, Part:BBa_K934001 (phaC1-A-B1) was rehydrated and transformed into our chassis so that PHB was produced and preliminary secretion assays could be performed before the Synthesis subgroup had completed their cloning.
Materials
  • iGEM 2017 distribution kit
  • ddH₂O
Protocols
  1. Add 10μL of ddH₂O to the desired well.
  2. Pipette up and down 3-5 times.
  3. Incubate at room temperature for 10 minutes.
  4. Transform cells with 1μL of rehydrated DNA. Store the remaining amount at -20°C.