Team:Amsterdam/Notebook/Protocols/Making electrocompetent Cells

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* Transform 50 μl of cells with 10 pg of standard pUC19
* Transform 50 μl of cells with 10 pg of standard pUC19
** This is 1 μl of standard pUC19 Invitrogen plasmid
** This is 1 μl of standard pUC19 Invitrogen plasmid
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* Hold on ice for 30 minutes
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* Heat shock 60 sec at 42°C
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* Add 200 μl [[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P3.)|SOC]]
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* shake at 37°C for 2 hours in 14 ml aerated tubes
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* Plate 20 μl and 200 on AMP plates using a sterile plate spreader
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** Transformation efficiency is number of colonies / (ng of DNA x partition of total volume plated) x 1000
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** Competence should be at least 5x10<sup>7</sup>. The higher the better.
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{{:Team:Amsterdam/Footer}}

Revision as of 09:38, 21 September 2011

Contents

Preparation of electrocompetent cells

Overview

We use a protocol commonly used in our host lab provided by Diewertje Piebes. The main advantage of electrocompetent cells compared to chemically competent cells are a higher level of competence (1-2 log higher). The disadvantage, though, is the high price of electroporation cuvettes and the implications this has on maximum number of transformations that can be performed in a single experiment.

Materials

  • Prechilled detergent-free, sterile glassware and plasticware. We use dedicated centrifuge tubes that are only used for preparation of competent cells
  • Table-top OD600nm spectrophotometer
  • SOB WITHOUT MgCl
  • 10% glycerol. Sterilise by passing through 0.22-µm filter. Store at 4°C

Procedure

  • Prepare TOP10 preculture by inoculating 3ml of LB medium with a -80°C TOP10 glycerol stock into and shake overnight at 37°C
  • Inoculate 200 ml of SOB containing no magnesium with 3 ml of preculture and grow in a 37°C shaker to an OD600 of 0.6-0.8
    • please note that E. coli growth rate is significantly reduced due to the absence of Magnesium
    • it is possible to add more preculture if you are in a hurry, however, it is not recommended
    • Prewarmed medium can shorten preparation time by up to one hour
  • Centrifuge at 4000g at 4°C for 10 minutes in a flat bottom centrifuge bottle
  • Note that from this point on the cells, media, bottles and glassware will have to be kept on ice at all times!! If possible it is recommended to work in a cold room.
  • decant supernatant and resuspend pellet in 50-100 ml of ice-cold 10% glycerol
  • Centrifuge at 4000g at 4°C for 10 minutes in a flat bottom centrifuge bottle
  • Once again decant supernatant and resuspend pellet in 50-100 ml of ice-cold 10% glycerol
    • This step can be repeated up to 3 times. Repeated cycles can increase competence, but will decrease yield
  • After one final centrifugation step resuspend cells in (2ml???) of ice-cold 10% glycerol
  • Aliquot 50 μl samples to sterile, prechilled, 1.5 ml or 2 ml eppendorf tubes and flash freeze by immersion in liquid nitrogen
  • Store at -80°C indefinitely
  • Test competence (see below)

Measurement of competence

  • Transform 50 μl of cells with 10 pg of standard pUC19
    • This is 1 μl of standard pUC19 Invitrogen plasmid



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