I am currently working on a CRISPR Cas9 project that involves preparing DH5a competent cells for bacterial transformation. I conducted heat-shock transformations using PUC19 as a positive control and my samples. In the case of PUC19, I used 2.5 µl with 50 µl of DH5a, while for my samples, I performed the heat-shock transformation twice. Initially, I used 1 µl with 150 µl of DH5a, and in the second attempt, I used 2 µl with 200 µl of DH5a. Unfortunately, I did not observe any colonies in both the positive control and my samples. However, after two nights, I noticed a few colonies only on the PUC19 plate.

I have concerns regarding the DH5a preparation. Firstly, the OD reached 0.569 before diluting it to the optimal density of 0.4. Is that correct to dilute?

Secondly, the ampicillin used had been stored at 4°C for almost 9 months, raising doubts about its effectiveness. Despite this, the absence of bacterial growth without ampicillin indicates that the ampicillin is still active.

I am seeking advice and opinions from experts on this matter. Any suggestions on optimizing DH5a preparation, troubleshooting the lack of colony formation in my samples, and ensuring the effectiveness of the stored ampicillin would be greatly appreciated.