Team:Tuebingen/Notebook/Protocols/chemo-competentcells

From 2013.igem.org

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   <li>Centrifuge cells at 2500 g and 4 °C for 10 min. Discard supernatant <u>completely</u>.</li>
   <li>Centrifuge cells at 2500 g and 4 °C for 10 min. Discard supernatant <u>completely</u>.</li>
   <li>Repeat the previous two steps.</li>
   <li>Repeat the previous two steps.</li>
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   <li><u>Completely</u> resuspend cells in 8 mL Inoue buffer at 0 °C. Add 1.5 mL DMSO and incubate for 10 min.</li>
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   <li><u>Completely</u> resuspend cells in 8 mL <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/inoue">Inoue buffer</a> at 0 °C. Add 1.5 mL DMSO and incubate for 10 min.</li>
   <li>Aliquot cells à 100 µL in Eppendorf-tubes and freeze in liquid nitrogen. Store at -80 °C.</li>
   <li>Aliquot cells à 100 µL in Eppendorf-tubes and freeze in liquid nitrogen. Store at -80 °C.</li>
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</ol>

Revision as of 23:51, 3 October 2013

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Chemo-competent E. coli cells
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Procedure

General advice:
It is advantageous to scale up the volume in order to yield more aliquots! Use additional flasks when necessary. Follow this protocol closely in order to yield best results.

  1. Inoculate 25 mL SOB-medium with one single colony from a fresh plate.
  2. Incubate for 8 h at 37 °C and 250 rpm.
  3. Inoculate three falcon tubes containing 100 mL SOB-medium each with 1 mL, 2 mL and 4 mL of the prepared pre-culture (i.e. 100 mL SOB + 1 mL pre-culture; 100 mL SOB + 2 mL pre-culture, and 100 mL SOB + 4 mL pre-culture).
  4. Incubate over night at 18 to 22 °C and 200 rpm.
  5. On the next day, check OD600. At OD600 = 0.55, put culture on ice for 10 min.
  6. Centrifuge cells at 2500 g and 4 °C for 10 min. Discard supernatant completely.
  7. Completely resuspend cell pellet in 30 mL Inoue buffer at 0 °C.
  8. Centrifuge cells at 2500 g and 4 °C for 10 min. Discard supernatant completely.
  9. Repeat the previous two steps.
  10. Completely resuspend cells in 8 mL Inoue buffer at 0 °C. Add 1.5 mL DMSO and incubate for 10 min.
  11. Aliquot cells à 100 µL in Eppendorf-tubes and freeze in liquid nitrogen. Store at -80 °C.


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