Team:DTU-Denmark/Competent cells

From 2011.igem.org

(Difference between revisions)
Line 2: Line 2:
Day 1:
Day 1:
-
# Inoculate 5 ml of LB from a colony or from a -80\^{o}C stock.
+
# Inoculate 5 ml of LB from a colony or from a -80<sup>o</sup>C stock.
Day 2:
Day 2:
# Dilute exponentially growing cells to OD600=0.05 and grow cells in a shaker to OD600=0.5-0.6 in pre-warmed LB. Remove the cells from the shaker and place on ice. Transfer liquid cultures to centrifuge tubes.
# Dilute exponentially growing cells to OD600=0.05 and grow cells in a shaker to OD600=0.5-0.6 in pre-warmed LB. Remove the cells from the shaker and place on ice. Transfer liquid cultures to centrifuge tubes.
Note: It’s very important to keep the cell on ice from now on.
Note: It’s very important to keep the cell on ice from now on.
# Centrifuge at 6 krpm for 10min; discard supernatant.
# Centrifuge at 6 krpm for 10min; discard supernatant.
-
# Gently resuspend in 5-7 ml of ice-cold 10% glycerol; consolidate to half the number of centrifuge tibes.
+
# Gently resuspend in 5-7 ml of ice-cold 10% glycerol; consolidate to half the number of centrifuge tubes.
# Fill up with 10% glycerol and centrifuge 10 min at 6 krpm.
# Fill up with 10% glycerol and centrifuge 10 min at 6 krpm.
# Repeat step 3 and 4 (no consolidation) two to three times.
# Repeat step 3 and 4 (no consolidation) two to three times.
# Resuspend in 5-7 ml of 10% glycerol and move to 15 ml or 50 ml tubes.
# Resuspend in 5-7 ml of 10% glycerol and move to 15 ml or 50 ml tubes.
# Centrifuge at 5 krpm for 5 minutes, discard supernatant and resuspend gently in about 2 ml 10% glycerol pro L culture.
# Centrifuge at 5 krpm for 5 minutes, discard supernatant and resuspend gently in about 2 ml 10% glycerol pro L culture.
-
# Flash freeze into Eppies placed in -80oC pure ethanol bath and store at -80oC. Make aliquots of 85µl, 135 µl and 175 µl for 2, 3 and 4 electroporations respectively.
+
# Flash freeze into Eppies placed in -80<sup>o</sup>C pure ethanol bath and store at -80oC. Make aliquots of 85µl, 135 µl and 175 µl for 2, 3 and 4 electroporations respectively.
Day 3:
Day 3:
# Transform cells with 1 µl of 10 pg/µl of pUC19 (AmpR) to test efficiency of the competent cells.
# Transform cells with 1 µl of 10 pg/µl of pUC19 (AmpR) to test efficiency of the competent cells.

Revision as of 14:39, 18 September 2011

Preparation of competent cells

Day 1:

  1. Inoculate 5 ml of LB from a colony or from a -80oC stock.

Day 2:

  1. Dilute exponentially growing cells to OD600=0.05 and grow cells in a shaker to OD600=0.5-0.6 in pre-warmed LB. Remove the cells from the shaker and place on ice. Transfer liquid cultures to centrifuge tubes.

Note: It’s very important to keep the cell on ice from now on.

  1. Centrifuge at 6 krpm for 10min; discard supernatant.
  2. Gently resuspend in 5-7 ml of ice-cold 10% glycerol; consolidate to half the number of centrifuge tubes.
  3. Fill up with 10% glycerol and centrifuge 10 min at 6 krpm.
  4. Repeat step 3 and 4 (no consolidation) two to three times.
  5. Resuspend in 5-7 ml of 10% glycerol and move to 15 ml or 50 ml tubes.
  6. Centrifuge at 5 krpm for 5 minutes, discard supernatant and resuspend gently in about 2 ml 10% glycerol pro L culture.
  7. Flash freeze into Eppies placed in -80oC pure ethanol bath and store at -80oC. Make aliquots of 85µl, 135 µl and 175 µl for 2, 3 and 4 electroporations respectively.

Day 3:

  1. Transform cells with 1 µl of 10 pg/µl of pUC19 (AmpR) to test efficiency of the competent cells.
  2. Plate 10 µl and 100 µl on LB+Amp (100 µg/ml) plates.

Day 4:

  1. Count colonies and calculate the transformation efficiency as colonies/µg of pUC DNA.
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