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Team:Macquarie Australia/WetLab/Protocols/CompetentCells
From 2014.igem.org
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<ul style="list-style-type: decimal;"> | <ul style="list-style-type: decimal;"> | ||
| - | <li>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- | + | <li>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 18-22<sup>o</sup>C, 200-250rpm.</li> |
<li>A600 should be 0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~ 0.5.</li> | <li>A600 should be 0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~ 0.5.</li> | ||
<li>Remove the flask from the incubator and place on ice for 10 minutes.</li> | <li>Remove the flask from the incubator and place on ice for 10 minutes.</li> | ||
| - | <li>Transfer the culture to a 15mL centrifuge tube and spin at 2500 x g for 10 min at | + | <li>Transfer the culture to a 15mL centrifuge tube and spin at 2500 x g for 10 min at 4<sup>o</sup>C</li> |
<li>Pour off and discard the supernatant, and immediately place the tube on ice.</li> | <li>Pour off and discard the supernatant, and immediately place the tube on ice.</li> | ||
<li>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> | <li>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> | ||
<li>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> | <li>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> | ||
<li>Incubate the tube on ice for 10 minutes.</li> | <li>Incubate the tube on ice for 10 minutes.</li> | ||
| - | <li>Centrifuge at 2,500 x g for 7 minutes at | + | <li>Centrifuge at 2,500 x g for 7 minutes at 4<sup>o</sup>C, discard the supernatant.</li> |
<li>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> | <li>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> | ||
<li>Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension, keeping the remainder on ice in the 15mL tube.</li> | <li>Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension, keeping the remainder on ice in the 15mL tube.</li> | ||
<li>Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice.</li> | <li>Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice.</li> | ||
<li>Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen.</li> | <li>Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen.</li> | ||
| - | <li>Repeat step 11-13 for the rest of your cell suspension in step 10. Store cells at - | + | <li>Repeat step 11-13 for the rest of your cell suspension in step 10. Store cells at -80<sup>o</sup>C. </li> |
</ul> | </ul> | ||
Revision as of 04:57, 16 October 2014
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 -80oC.
