Shivani Soni first, please give more information about your protocol

1. How many volume of bacteria cell culture you used for plasmid isolation? and how long the culture incubation?

2. no precipitation could happen for several reason

• Incorrect Preparation of BuffersProblem: P3 buffer may not be prepared correctly, especially if the potassium acetate or acetic acid concentrations are too low. Solution: Double-check the P3 buffer preparation. Ensure the final solution has ~3 M potassium acetate and is adjusted to pH ~5.2 with acetic acid.
• Insufficient Lysis with P2 BufferProblem: Incomplete lysis of cells due to insufficient SDS or NaOH in P2 buffer, or if the lysis step was too short. Solution: Ensure proper preparation of P2 buffer. Incubate for 2–5 minutes after adding P2 to allow full lysis but avoid over-incubation (no more than 5 minutes) to prevent DNA degradation. Over-incubation with P2 BufferProblem: If cells remain in P2 buffer for too long, plasmid and genomic DNA may degrade, or the solution may not neutralize properly. Solution: Add P3 immediately after the lysis step to neutralize the alkaline conditions. Low Cell Density or Poor Cell PelletProblem: Insufficient starting material means there isn’t enough genomic DNA or debris to form a visible precipitate. Solution: Increase the starting cell culture volume or pellet cells more effectively during the initial centrifugation step.

If you do not see any precipitation at all, the problem is with your kit components. Upon adding P3 buffer (potassium acetate) to P1+P2 mix (contains SDS), you are supposed to see massive precipitation of poorly soluble potassium-DS even in the absence of cells / proteins / DNA etc in the input. You may try mixing equal volumes of P2 and P3, and see if precipitation occurs or not. No precipitation might be due to SDS depletion in P2 solution. Upon prolonged storage at alkaline pH, SDS tends to decompose to sodium sulfate and dodecane, which would make the solutionr unusable for plasmid purification. When P2 is stored in the cold, SDS may precipitate and sit at the bottom, which makes its concentration in the upper part of the vial insufficient.

Is anyone else in your group using the same kit and having the same issue? If so, that would indicate a kit problem. Kit reagents can expire (or go off/bad due to improper storage etc.). Are you sure the P3 is from that kit? I ask my lab to date all kit bottles (and the box) just in case someone grabbed the older backup supplies.

Thank you very much, everyone. I got the protein precipitation. I am highly grateful for all your suggestions. I am looking for your help in the future as well.

What did you end up doing that fixed the issue?

I changed all the buffers and used the kit one based as well. It worked well then