Likely there was high ionic strength in your preparation (have you seen an arc?).

What goes into your preparation? Cells and DNA. Either your treatment of cells was too harsh and caused lysis for some reason, or your DNA contains salts. If you pulse only the cells, how does it work? How did you prepare your DNA?

Hello,

Generally, the problem in electroporation is the presence of salt, which will prevent the electric current from passing and crossing the tank.

Try as he suggested to you Tomáš Hluska, with cells without the presence of DNA to see if indeed the problem comes from your DNA.

If so, purify your DNA by precipitation and resuspension. Alternatively, prepare competent for heat shock instead of electroporation.

Thank you Tomáš Hluska and Sofiane Benyamina

I seemed to have centrifuged the cells at a higher RPM, or salts remained even after washing.

I prepared Electrocompetent cells again; washing was done with 10% glycerol. The time constant with only cells was 5.9, and with plasmid cells mixture was 5.3.

I am facing another problem now: my control plate with only cells shows the same growth as the experimental plate. I am using Ampicillin plates with 100ug/ml concentration. The cells are WT K-12 and should not show growth on Amp plates.

Hello,

If your host cell without the vector should not grow in the presence of the antibiotic (Have you ever tested before that your bacterium is sensitive to this antibiotic?), maybe the problem lies in the antibiotic itself.

Is it functional? is it not expired? kept in good condition?

Also, is your control culture not contaminated?

Hi Shilpi Kumari ,

I'm glad we could help.

Your next question is a tough one. As Sofiane suggested, it may be either malfunctioning antibiotics or resistant culture.

For that, I'd try to get some independent ampicillin (from another laboratory, or open brand new bottle) and prepare two sets of plates - ones with your Amp (A), ones with the other (B). You can even use higher concentration of the other Amp to make sure it's working.

Then I would try to get some cells, which should be 100% sensitive. Again, if you can get them from another laboratory, that'd be great. If not, maybe try some bacteria with another plasmid resistant e.g. to Kan or any other antibiotics. That's the first. Second would be your cells before transformation. Third would be your control transformation cells. Fourth would be cells transformed with your plasmid.

Then, you would combine them to get all options (1A, 2A, 3A, 4A, 1B, 2B, 3B, 4B). And you will see what will be the pattern.

BTW if all the cells were resistant, your plates should be covered with bacteria (unless you dilute them alot). If you get colonies, only some of them are resistant. That would suggest rather contamination during the transformation process.

(in that case, to simplify and accelerate things, you may just try to plate the cells before transformation on your plate, if you have both left)

Electroporation of E. coli cells involves the application of electrical pulses to the cells to introduce foreign DNA or other molecules into the cell. The time constant in this context refers to the time it takes for the electric field to dissipate within the cells.

A time constant of 2.2ms after electroporation of E. coli cells suggests that the cells are tolerating the electrical pulses without significant damage. However, the time constant may vary depending on the specific conditions of the electroporation, such as the intensity and duration of the pulses. Further experimentation may be necessary to fully understand the relationship between electroporation conditions and time constants in E. coli cells.