It is possible that the ligation buffer could decrease the efficiency of electroporation in E. coli cells. There are several potential reasons for this.

One possibility is that the components of the ligation buffer, such as the concentrations of Mg2+ ions and divalent cations, could affect the electroporation efficiency. These ions can influence the stability and integrity of the bacterial cell membrane, and high concentrations of them can cause the cell membrane to become less permeable to DNA. This could potentially reduce the efficiency of electroporation.

Another possibility is that the presence of the ligation buffer could alter the properties of the DNA itself, such as its charge or conformation. This could also potentially affect the efficiency of electroporation.

It is also possible that there could be other factors contributing to the decreased efficiency of electroporation with the ligation buffer. For example, the size of the plasmid (10 kb) could be a factor, as you mentioned. Large plasmids may be more difficult to transform than smaller ones due to their size and complexity. Additionally, the quality and purity of the DNA could also potentially affect the efficiency of transformation.

To troubleshoot this issue, it may be helpful to try adjusting the conditions of the electroporation, such as the voltage and pulse duration, or to try using a different electroporation buffer or protocol. It may also be helpful to try purifying the DNA to remove any contaminants or impurities that could be affecting the transformation efficiency.

Probably not a significant factor. But you could always do a cleanup of your ligated plasmid + insert.

A much more reasonable explanation is that doing a digest, then ligation, then transformation means you are simply adding in fewer plasmid molecules.

Look, I'm assuming the goal is to clone something into a plasmid and then use that plasmid as part of an experiment. Your goal is NOT to optimize the transformation, just have working well enough that you get some colonies to test. Spread more cells on your selection plates and call it good.

Good luck!

Hi

The observations are normal. Digestion and re-ligation will decrease the efficiency to certain extent, and this is not due to direct impact of transformation but also due to the fact that all the plasmid molecules may not be able to ligate. Also the ions in the ligation buffer do reduce the transformation efficiency as the impact the time-constant of the electric pulse. If the aim is not to generate a big library, try to optimize the protocol a little bit and get the clones.

Dear Antonia

theoretically is possible since when we prepare the competent cells for elettroporation the cells have to be removed from salts performing several wash in milliQ water since salts camight cause an electrical discharge (known as arcing), which often reduces the viability of the bacteria.

You can skip this problem or performing trasformation with chemically competent cells of perfroming a dialisys of your ligase mixture using the millipore membrane filters

https://www.merckmillipore.com/IT/it/product/MF-Millipore-Membrane-Filters,MM_NF-C152#specifications

i have done this in the past using the 0.025um pore size folllowing the same protocoll

1) fill a 6 well plate with 2ml of milliQ water for each well

2) Put a filter disc on the surface of the water

3) Add 10ul of ligase reaction on the surface of the filrter disc

4) close the plate lide

5) incubate at room temperature an wait at least 1h

6) recover the ligase reaction and perform the elettroporation.

i hope this could be usefull to you

best regards

Manuele

It is generally recommended to do phenol-chloroform purification of ligation mixture before electroporation (this is not needed if you do heat-shock).