Difference between revisions of "Team:Calgary/Protocols"

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                                     <p>Overnight culture of desired bacteria</p>  
 
                                     <p>Overnight culture of desired bacteria</p>  
 
                                   <p>70% ethanol</p>
 
                                   <p>70% ethanol</p>
−
                                   <p>Spreading rod</p>
+
                                   <p>Spreading rod</p>                                
−
                                  <p>Bunsen burner</p>
+
 
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                         <i id="icon5" class="fas fa-plus accordion-icon" aria-hidden="true"></i>
 
                         <h3>
 
                         <h3>
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                             Protocol 5
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                             Preparation of Chemically Competent <i>Escherichia coli</i> Cells
 
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                                 </td>
 
                                 <td>
 
                                 <td>
−
                                     <p>Luria-Bertani agar plate with appropriate antibiotic (if required)</p>
+
                                    <p><i>Escherichia coli</i> DH5α cells</p>
−
                                    <p>Overnight culture of desired bacteria</p>  
+
                                     <p>Luria-Bertani broth</p>  
−
                                   <p>70% ethanol</p>
+
                                  <p>125mm culture tubes</p>
−
                                   <p>Spreading rod</p>
+
                                   <p>250mL Erlenmeyer flasks</p>
−
                                   <p>Bunsen burner</p>
+
                                   <p>50mL Falcon tubes, pre-chilled</p>
 +
                                   <p>1M KCl</p>
 +
                                  <p>1M MgSO<sub>4</sub></p>
 
                                 </td>
 
                                 </td>
 
                             </tr>
 
                             </tr>

Revision as of 23:59, 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

Materials

Luria-Bertani agar plate with appropriate antibiotic (if required)

Overnight culture of desired bacteria

70% ethanol

Spreading rod
Protocol
  1. Using aseptic technique, pipette 100μL of bacterial culture onto agar plate
  2. Dip spreading rod in 70% ethanol, pass over flame, and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture
  3. Use rod to spread bacterial culture over entire plate, spinning the plate at the same time
  4. Dip spreading rod in 70% ethanol, pass over flame, and allow excess liquid to burn off
  5. Incubate plates at 37°C overnight or until growth is observed

Materials

Escherichia coli DH5α cells

Luria-Bertani broth

125mm culture tubes

250mL Erlenmeyer flasks

50mL Falcon tubes, pre-chilled

1M KCl

1M MgSO4
Protocol
  1. Using aseptic technique, pipette 100μL of bacterial culture onto agar plate
  2. Dip spreading rod in 70% ethanol, pass over flame, and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture
  3. Use rod to spread bacterial culture over entire plate, spinning the plate at the same time
  4. Dip spreading rod in 70% ethanol, pass over flame, and allow excess liquid to burn off
  5. Incubate plates at 37°C overnight or until growth is observed

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.