Difference between revisions of "Team:Toulouse/Experiments"

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         <li><a href="#part6">- Digestion: Parts have the same antibiotic resistance</a></li>
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         <li><a href="#part7">- Gel extraction</a></li>
 
         <li><a href="#part8">- Ligation</a></li>
 
         <li><a href="#part8">- Ligation</a></li>
 
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<li><a href="#part9">- Transformation</a></li>
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<h3 style="font-size:20px;">Both parts have the same antibiotic resistance</h3>
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<h3>Digestion</h3>
 
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<h3 style="font-size:20px;">Both parts have the same antibiotic resistance</h3>
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             <td><p>5 µL of miniprep plasmid</p></td>
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             <td><p>1 µg of </p></td>
 
             <td><p>10 µL of miniprep plasmid</p></td>
 
             <td><p>10 µL of miniprep plasmid</p></td>
 
          
 
          
 
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             <td><p>2 µL of each restriction enzymes</p></td>
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             <td><p>1 µL of each restriction enzymes</p></td>
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             <td><p>2 µL of each restriction enzymes</p></td>
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             <td><p>1 µL of each restriction enzymes</p></td>
 
            
 
            
 
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<h3>Cloning</h3>
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<h3>Gel extraction</h3>
 
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Revision as of 13:53, 1 September 2015

iGEM Toulouse 2015

Experiments & Protocols


Transformation Protocol: RbCl method

Cloning

Transformation Protocol: RbCl method

Media and solution

YETM 500mL TFB1 200mL TFB2 200mL
  • 2.5g Yeast Extract
  • 10g Tryptone
  • 5g MgSO4.7H2O
  • Adjust pH to 7.5 with KOH
  • For Plates: add 7.5g of Agar
  • 0.59g KOAc
  • 2.42g RbCl
  • 0.29g CaCl2.2H2O
  • 1.98g MnCl2.4H2O
  • Adjust to pH 5.8 with 0.2 M acetic acid
  • Add dH2O to 200mL
  • Filter sterilize
  • Store refrigerated at 4°C
  • 0.42g MOPS
  • 2.21g CaCl2.2H20
  • 0.24g RbCl
  • 30g Glycerol
  • Adjust to pH 6.5 with KOH
  • Add dH2O to 200mL
  • Filter sterilize
  • Store refrigerated at 4°C

Preparation of Competent Cells

  • 1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37°C
  • 2. Pick a single fresh colony to inoculate 5mL of YETM medium. Grow over night at 37°C.
  • Do not vortex cells at any time after this point in the procedure
  • 3. Dilute 1mL of culture into 50mL YETM medium prewarmed to 37°C
    • Grow at 37°C for 2hr with agitation
    • Volumes can be scaled up 5X and all of the 5mL overnight culture can be used
  • 4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes
  • 5. Centrifuge for 10 minutes at 2000rpm at 4°C. Immediately aspirate off all of the supernatant
  • Do not allow cells to warm above 4°C at any time in this procedure
  • 6. Resuspend cells in 10mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette
  • 7. Incubate cells on ice for 5min
  • 8. Repeat step 5
  • 9. Resuspend cells in 2mL of ice-cold TFB2 with gentle re-pipetting. Use micropipet tip (plastic)
  • 10. Incubate cells on ice for 15 minutes
  • Cells may be used for transformation or frozen
    • To freeze: aliquot cell in 200μL volumes into prechilled 0.5mL microfuge tube (on ice)
    • Freeze immediately in liquid nitrogen
    • Store cells frozen at -80°C

Transformation of Competent Cells

  • 1. If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed.
    Immedately place on ice for about 5 minutes
  • 2. Add to 1,5mL eppendorff on ice: 2-3μL iGEM plate or 1μL plasmid or 10μL ligation.
  • 3. Add 100μL of competent cells and mix by gentle re-pipetting
  • 4. Incubate cells on ice for 20-30 minutes
  • 5. Heat shock the cells exactly 90 seconds at 42°C
  • 6. Return cells on ice for 2 minutes
  • 7. Add 1mL of YETM medium. Incubate at 37°C for 45-60 minutes with slow gentle shaking
  • 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate overnight at 37°C

Minipreps

  • 1. Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)
  • 2. Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic
  • 3. Let the culture grow overnight at 37°C in a shaking incubator
  • 4. Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55°C
  • 5. Keep the tubes at -20°C

Cloning

Digestion

Both parts have the same antibiotic resistance

Vector Insert

1 µg of

10 µL of miniprep plasmid

1 µL of each restriction enzymes

1 µL of each restriction enzymes

2 µL of Green Buffer

2 µL of Green Buffer

9 µL of Milli-Q water

4 µL of Milli-Q water

Incubate 15 minutes at 37°C

Gel extraction

References