Fong:Electrotransformation

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==Overview==
==Overview==

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Overview

This protocol describes an effective method for transforming electrocompetent E. coli cells with DNA. It is important to carefully manage temperature throughout this procedure, so be sure to chill all cuvettes and microcentrifuge tubes on ice prior to and during use. Perform all centrifugation steps with the centrifuge in rm 407 set at 0°C.

Materials

  • 50μL electrocompetent E. coli cells per sample
  • 1-2μL DNA
  • Microcentrifuge tubes for mixing
  • Electrotransformation cuvette (2mm)
  • BioRad MicroPulser
  • Ice
  • Micropipettes
  • SOC (or LB medium) for recovery
  • Selective LB medium, in plates and liquid form
  • Plate spreader
  • Sterile loop
  • 30-40% sterile glycerol and cryovials

Procedure

  1. Remove electrocompetent E. coli cells from the -80°C freezer and lay across ice to thaw. This step can be expedited by thawing carefully in your hand, but care must be taken to make sure that the entire culture remains chilled at all times. Make note of the date on which the electrocompetent cells were prepared.
    • We generally freeze electrocompetent cells into 250μL aliquots, each good for 5 samples. Be sure to plan for negative controls which are pulsed without added DNA and positive controls which are plated on non-selective medium to rule out bad antibiotics or non-viable cells.
  2. Place sterile microcentrifuge tubes on ice for mixing cells and DNA.
  3. Pipette 50μL of cell suspension into a chilled tube.
  4. Add 1-2 μL of DNA (1μL works fine for DNA from the BioBricks distribution plates) to the 50μL sample, mix carefully by pipette and wait about a minute.
  5. Transfer the mixture to a chilled electrotransformation cuvette, tap lightly to get mixture to the bottom of the cuvette, and pulse the cuvette on the Ec2 setting (for 2mm cuvettes).
  6. Immediately add 1mL of SOC medium (LB can also work here in a pinch, but SOC is more accommodating to the shocked cells) and mix quickly but gently by pippette, and incubate at 37°C for 30-60min.
    • NB: the manual for the gene pulser indicates that it is critical to add the recovery medium as quickly as possible here, as delaying this step by even 1 min decreases efficiency by 3 fold.
  7. Spread 35μL onto a warmed selective LB plate and incubate overnight, or longer if necessary.
  8. After incubation, pick isolated colonies and transfer to 5mL warmed liquid LB selective medium and incubate overnight (< 12hr).
  9. Freeze a glycerol stock of the transformed cells and perform QIAGEN MiniPrep at this step as needed.
    • Be sure to label stocks with gene name and plasmid info along with standard date and initials.

Notes

References

This protocol is based on the SOP from the Palsson Lab at UCSD which is in turn a modification of Dr. Datsenko's method.

Contact

Chris or George


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