Difference between revisions of "Team:Toulouse/Experiments"

Line 192: Line 192:
 
<li> 7. Add 1mL of YETM medium. Incubate at 37°C for 45-60 minutes with slow gentle shaking </li>
 
<li> 7. Add 1mL of YETM medium. Incubate at 37°C for 45-60 minutes with slow gentle shaking </li>
 
<li> 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate  overnight at 37°C</li>
 
<li> 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate  overnight at 37°C</li>
−
 
+
</ul>
 
</div> <!-- COMPETENT CELLS END -->
 
</div> <!-- COMPETENT CELLS END -->
  
Line 217: Line 217:
  
 
<div class="subtitle" >    
 
<div class="subtitle" >    
−
<h3>Digestion</h3>
+
<h3>First step: Digestion</h3>
 
</div>
 
</div>
  
Line 228: Line 228:
 
         <thead>
 
         <thead>
 
           <tr>
 
           <tr>
−
             <th>Vector</th>
+
             <th>Both parts</th>
−
            <th>Insert</th>
+
 
           </tr>
 
           </tr>
 
         </thead>
 
         </thead>
Line 263: Line 262:
 
   
 
   
 
<center><div class="subtitle" >    
 
<center><div class="subtitle" >    
−
<h3>Gel extraction</h3>
+
<h3 style="font-size:20px;">The two parts have a different antibiotic resistance</h3>
 
</div></center>
 
</div></center>
  
 +
 +
<div class="group center">
 +
<table class="df" style="font-size:15px;">
 +
        <thead>
 +
          <tr>
 +
            <th>Both parts</th>
 +
          </tr>
 +
        </thead>
 +
        <tbody>
 +
          <tr>
 +
            <td><p>5 µL of miniprep plasmid</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>1 µL of each restriction enzymes</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>2 µL of Green Buffer</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>9 µL of Milli-Q water</p></td>
 +
          </tr>
 +
  <tr>
 +
  <td><p>Incubate 15 minutes at 37°C</p></td>
 +
  </tr>
 +
        </tbody>
 +
      </table>
 +
  </div>
 +
 
 +
<div class="subtitle" >  
 +
<h3>Second step: Ligation</h3>
 +
</div> 
 +
 
 +
<div class="group center">
 +
<table class="df" style="font-size:15px;">
 +
        <thead>
 +
          <tr>
 +
            <th>Mix</th>
 +
<th>Control</th>
 +
          </tr>
 +
        </thead>
 +
        <tbody>
 +
          <tr>
 +
            <td><p>10 µL of insert</p></td>
 +
<td><p>no insert</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>4 µL of vector</p></td>
 +
<td><p>4 µL of vector</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>2 µL of 10x T4 buffer</p></td>
 +
<td><p>2 µL of 10x T4 buffer</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>0.5 µL of T4 ligase</p></td>
 +
<td><p>0.5 µL of T4 ligase</p></td>
 +
          </tr>
 +
  <tr>
 +
  <td><p>3.5 µL of Milli-Q water</p></td>
 +
  <td><p>13.5 µL of Milli-Q water</p></td>
 +
  </tr>
 +
        </tbody>
 +
      </table>
 +
  </div>
 +
 
 
   </main>
 
   </main>
 
</div>
 
</div>

Revision as of 14:46, 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

First step: Digestion

Both parts have the same antibiotic resistance

Both parts

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

The two parts have a different antibiotic resistance

Both parts

5 µL of miniprep plasmid

1 µL of each restriction enzymes

2 µL of Green Buffer

9 µL of Milli-Q water

Incubate 15 minutes at 37°C

Second step: Ligation

Mix Control

10 µL of insert

no insert

4 µL of vector

4 µL of vector

2 µL of 10x T4 buffer

2 µL of 10x T4 buffer

0.5 µL of T4 ligase

0.5 µL of T4 ligase

3.5 µL of Milli-Q water

13.5 µL of Milli-Q water

References