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| - | {{:Team:DTU-Denmark/Templates/Standard_page_begin|Preparation of competent cells}}
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| - | == Preparation of competent cells ==
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| - | Day 1:
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| - | # Inoculate 5 ml of LB from a colony or from a -80<sup>o</sup>C stock.
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| - | Day 2:
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| - | # 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.
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| - | Note: It’s very important to keep the cell on ice from now on.
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| - | # Centrifuge at 6 krpm for 10min; discard supernatant.
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| - | # Gently resuspend in 5-7 ml of ice-cold 10% glycerol; consolidate to half the number of centrifuge tubes.
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| - | # Fill up with 10% glycerol and centrifuge 10 min at 6 krpm.
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| - | # Repeat step 3 and 4 (no consolidation) two to three times.
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| - | # Resuspend in 5-7 ml of 10% glycerol and move to 15 ml or 50 ml tubes.
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| - | # Centrifuge at 5 krpm for 5 minutes, discard supernatant and resuspend gently in about 2 ml 10% glycerol pro L culture.
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| - | # 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.
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| - | Day 3:
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| - | # Transform cells with 1 µl of 10 pg/µl of pUC19 (AmpR) to test efficiency of the competent cells.
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| - | # Plate 10 µl and 100 µl on LB+Amp (100 µg/ml) plates.
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| - | Day 4:
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| - | # Count colonies and calculate the transformation efficiency as colonies/µg of pUC DNA.
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Latest revision as of 19:09, 21 September 2011
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