Team:Groningen/protocols/Transformation EC

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<div class="mainContent">
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<h2>E. Coli transformation protocol</h2>
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<h2><i>E. coli</i> transformation protocol</h2>
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<h3>Materials:</h3>
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<h5>Materials:</h5>
<ul type="square">
<ul type="square">
<li>LB plates with selection markers (antibiotics, inducers,…)</li>
<li>LB plates with selection markers (antibiotics, inducers,…)</li>
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</ul>
</ul>
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<h3>Steps:</h3>
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<p>
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<h5>Reaction procedure:</h5>
<ol>
<ol>
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<li>Prepare plates with the correct selection marker (which are usually kept in the fridge or in the freezer depending on the stability of the molecule) for each ligation reaction.</li>
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<li>Prepare plates with the correct antibiotic selection marker.</li>
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<li>Centrifuge the tubes containing the ligation reactions to collect the contents at the bottom. Add the 5μl ligation reaction to a sterile (17 × 100mm) polypropylene tube or a 1.5ml microcentrifuge tube on ice (see Note 1).</li>
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<li>Prechill the eppendorf tubes on ice.</li>
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<li>Remove tube(s) of frozen Competent Cells from the storage (-80 °C freezer) and place in ice until just thawed (about 5 minutes). Mix the cells by gently flicking the tube. Avoid excessive pipetting, as the competent cells are extremely fragile.</li>
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<li>Remove a tube of frozen competent <i>E. coli </i>(DH5&#945;) cells from the storage (-80°C freezer) and thaw it on ice (about 5 minutes). Mix the cells by gently flicking the tube. Avoid excessive pipetting, as the competent cells are extremely fragile.</li>
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<li>Carefully transfer 50μl of cells into each tube prepared in Step 2.</li>
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<li>Carefully transfer 50 μl of cells into each tube prepared in Step 2.</li>
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<li>Gently flick the tubes to mix and place them on ice for 20 minutes.</li>
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<li>Add 10 μl ligation reaction to the competent cells.</li>
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<li>Heat-shock the cells for 80 seconds in a water bath at exactly 37°C (do not shake and make sure the content of the tube is all at the bottom).</li>
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<li>Gently flick the tubes to mix and place them on ice for 30 minutes.</li>
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<li>Heat-shock the cells for 60 seconds in a water bath at 42°C (do not shake and make sure the content of the tube is all at the bottom).</li>
<li>Immediately return the tubes to ice for 2 minutes.</li>
<li>Immediately return the tubes to ice for 2 minutes.</li>
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<li>Add 900μl room-temperature LB broth to the tubes containing cells.</li>
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<li>Add 1 ml LB broth (RT) to the reaction mixture.</li>
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<li>Incubate for 1.5 hours at 37°C with shaking (~150rpm).</li>
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<li>Incubate at 37°C for 1 hour with shaking (~150 rpm).</li>
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<li>Plate 200μl of each transformation culture on LB plates.</li>
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<li>Centrifuge the tubes at 14000 rpm for 1 min.</li>
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<li>Empty the tube until about 200 μl supernatant is left.</li>
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<li>Resuspend the pellet in the supernatant.</li>
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<li>Plate 200 μl of each transformation culture on LB agar plates with the correct resistant marker.</li>
<li>Incubate the plates overnight (16–24 hours) at 37°C.</li>
<li>Incubate the plates overnight (16–24 hours) at 37°C.</li>
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<li>Store of plates at 4°C (after 37°C overnight incubation).</li>
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<li>Afterwards the plates can be stored at 4°C.</li>
</ol>
</ol>

Latest revision as of 15:11, 27 September 2013

E. coli transformation protocol

Materials:
  • LB plates with selection markers (antibiotics, inducers,…)
  • LB broth
  • Eppendorf tubes (preferably 2 ml)
  • Spreaders
  • DNA to be transformed

Reaction procedure:
  1. Prepare plates with the correct antibiotic selection marker.
  2. Prechill the eppendorf tubes on ice.
  3. Remove a tube of frozen competent E. coli (DH5α) cells from the storage (-80°C freezer) and thaw it on ice (about 5 minutes). Mix the cells by gently flicking the tube. Avoid excessive pipetting, as the competent cells are extremely fragile.
  4. Carefully transfer 50 μl of cells into each tube prepared in Step 2.
  5. Add 10 μl ligation reaction to the competent cells.
  6. Gently flick the tubes to mix and place them on ice for 30 minutes.
  7. Heat-shock the cells for 60 seconds in a water bath at 42°C (do not shake and make sure the content of the tube is all at the bottom).
  8. Immediately return the tubes to ice for 2 minutes.
  9. Add 1 ml LB broth (RT) to the reaction mixture.
  10. Incubate at 37°C for 1 hour with shaking (~150 rpm).
  11. Centrifuge the tubes at 14000 rpm for 1 min.
  12. Empty the tube until about 200 μl supernatant is left.
  13. Resuspend the pellet in the supernatant.
  14. Plate 200 μl of each transformation culture on LB agar plates with the correct resistant marker.
  15. Incubate the plates overnight (16–24 hours) at 37°C.
  16. Afterwards the plates can be stored at 4°C.