Electroporation Questions

Electroporation Questions

Postby bioforum on Mon Jul 21, 2014 9:45 am

I have been trying to transfect a gene that I have placed in a pUC19 plasmid into electrocompetent e. Coli. Unfortunately, my cells seem to die shortly after transfection every time! What am I doing wrong?
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Re: Electroporation Questions

Postby ribo1221 on Mon Jul 21, 2014 11:44 am

I typically use this protocol (https://www.neb.com/protocols/1/01/01/electroporation-protocol-c2986) from New England Bio labs. We run it as follows.

1. Bring culture tubes with a round bottom (we use VWR #60818-667) to room temperature. Warm the SOC recovery medium to 37°C in a water bath. Warm your selection plates to 37°C for 1 hour.
2. Put 1 mm electroporation cuvettes (1mm) and microcentrifuge tubes for cells and DNA in ice bucket with ice.
3. For a transformation positive control, dilute the control pUC19 with sterile water by 1:5 to 10 pg/μl final concentration.
4. Thaw electrocompetent cells (we use the New England Bio labs turbo ones) on ice for approx 10 minutes and flick the tubes gently to mix the cells. Transfer 25 μl of the cells to one of the chilled microcentrifuge tubes from step 2 . Add 1 μl of your DNA solution to the chilled tube with the cells.
5. Transfer the mix of cells and DNA from the microfuge tube into a chilled electroporation cuvette, ensuring that there are no bubbles and the cells deposit on the bottom of the cuvette. Electroporate using the using the following settings: 2.1 kV, 100 Ω, and 25 μF. Our lab usually uses a time constant of ~2.6 ms.
6. Immediately after electroporation, pipette 975 µl of 37°C SOC (from step 1) to the cuvette, gently mixing the solution up and down twice, then transfer the contents of the cuvette to the culture tubes from step 1.
7. Shake the culture tubes vigorously at 250 rpm or rotate continuously for 1 hour at 37°C.
8. Dilute the cells to desired concentration and spread 100-200 μl cells onto a pre-warmed selection plate (from step 1).
9. Incubate selection plates 8 -12 hours at 37°C.
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Re: Electroporation Questions

Postby rtpcrman on Mon Jul 21, 2014 12:19 pm

NEB also offers some important tips you should make sure you're doing to increase post-electroporation cell viability: https://www.neb.com/tools-and-resources/usage-guidelines/electroporation-tips
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Re: Electroporation Questions

Postby submarine on Mon Jul 21, 2014 1:07 pm

Also you should make sure to dialyze your DNA into DI water- the salts in your buffers can cause problems with the electroporation process. A standard dialysis membrane from millipore works quite well for this purpose.
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