Team:Macquarie Australia/WetLab/Protocols/CompetentCells

From 2014.igem.org

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<h3>Making Competent Cells</h3>
<h3>Making Competent Cells</h3>
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1. Using a sterile plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate. Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22oC, 200-250rpm.
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Using a sterile plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate. Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22oC, 200-250rpm.
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2. A600 should be 0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~ 0.5.</li>
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A600 should be 0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~ 0.5.</li>
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3. Remove the flask from the incubator and place on ice for 10 minutes.</li>
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Remove the flask from the incubator and place on ice for 10 minutes.</li>
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4. Transfer the culture to a 15mL centrifuge tube and spin at 2500 x g for 10 min at 4oC</li>
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Transfer the culture to a 15mL centrifuge tube and spin at 2500 x g for 10 min at 4oC</li>
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5. Pour off and discard the supernatant, and immediately place the tube on ice.</li>
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Pour off and discard the supernatant, and immediately place the tube on ice.</li>
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6. Resuspend your cells in 1mL of ice-cold TB buffer, make sure there are no clumps of cells left, but also treat your cells gently and keep them cold.</li>
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Resuspend your cells in 1mL of ice-cold TB buffer, make sure there are no clumps of cells left, but also treat your cells gently and keep them cold.</li>
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7. Add ice-cold TB buffer to bring volume up to 1/5th of the original culture volume (~30mL in this case). Mix the tube by gently inverting 3 times.</li>
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Add ice-cold TB buffer to bring volume up to 1/5th of the original culture volume (~30mL in this case). Mix the tube by gently inverting 3 times.</li>
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8. Incubate the tube on ice for 10 minutes.</li>
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Incubate the tube on ice for 10 minutes.</li>
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9. Centrifuge at 2,500 x g for 7 minutes at 4oC, discard the supernatant.</li>
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Centrifuge at 2,500 x g for 7 minutes at 4oC, discard the supernatant.</li>
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10. Gently resuspend the cells in ~1/20th of the original culture volume of ice-cold TB buffer. NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. e.g. if the culture OD600 was 0.1 then resuspend in 1/100th of original volume.</li>
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Gently resuspend the cells in ~1/20th of the original culture volume of ice-cold TB buffer. NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. e.g. if the culture OD600 was 0.1 then resuspend in 1/100th of original volume.</li>
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11. Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension, keeping the remainder on ice in the 15mL tube.</li>
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Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension, keeping the remainder on ice in the 15mL tube.</li>
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12. Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice.</li>
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Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice.</li>
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13. Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen.</li>
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Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen.</li>
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14. Repeat step 11-13 for the rest of your cell suspension in step 10. Store cells at -80oC. </li>
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Repeat step 11-13 for the rest of your cell suspension in step 10. Store cells at -80C. </li>
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Revision as of 04:39, 16 October 2014

Protocols


Making Competent Cells

  • Using a sterile plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate. Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22oC, 200-250rpm.
  • A600 should be 0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~ 0.5.
  • Remove the flask from the incubator and place on ice for 10 minutes.
  • Transfer the culture to a 15mL centrifuge tube and spin at 2500 x g for 10 min at 4oC
  • Pour off and discard the supernatant, and immediately place the tube on ice.
  • Resuspend your cells in 1mL of ice-cold TB buffer, make sure there are no clumps of cells left, but also treat your cells gently and keep them cold.
  • Add ice-cold TB buffer to bring volume up to 1/5th of the original culture volume (~30mL in this case). Mix the tube by gently inverting 3 times.
  • Incubate the tube on ice for 10 minutes.
  • Centrifuge at 2,500 x g for 7 minutes at 4oC, discard the supernatant.
  • Gently resuspend the cells in ~1/20th of the original culture volume of ice-cold TB buffer. NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. e.g. if the culture OD600 was 0.1 then resuspend in 1/100th of original volume.
  • Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension, keeping the remainder on ice in the 15mL tube.
  • Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice.
  • Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen.
  • Repeat step 11-13 for the rest of your cell suspension in step 10. Store cells at -80C.

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