Plasmid Miniprep & Alkaline Lysis Troubleshooting Guide

Low plasmid yield

Low yield is the most frequent failure point in plasmid purification. It is often the result of a mismatch between biological input and chemical capacity.

Plasmid copy number

High-copy plasmids (e.g., pUC) can yield ≈20 μg per 3 mL of culture. Low-copy plasmids (e.g., pBR322, pET, or many cosmic vectors) may yield <2 μg from the same volume.

Fix: Verify the origin of replication (ori) of your vector. For low-copy plasmids, double the culture volume or use a "Midi" scale kit.

Incomplete cell resuspension

If the bacterial pellet is not a homogenous slurry before adding Buffer P2, lysis will be localized and inefficient, leaving DNA trapped in unlysed clumps.

Fix: Ensure no visible clumps remain after adding Buffer P1. Vortexing is safe only at this specific step.

SDS precipitation (Buffer P2)

Buffer P2 contains Sodium Dodecyl Sulfate (SDS), which precipitates out of solution at temperatures below 20°C. If the buffer is cloudy, the concentration of active detergent is too low to effectively solubilize the cell membrane.

Fix: Inspect Buffer P2 for white precipitates. If present, warm the bottle in a 37°C water bath until completely clear before use.

Column overloading

Excessive culture volume (> 5 mL) or high-density media (like TB) creates a lysate too viscous for the silica membrane. This prevents efficient DNA binding and can lead to column clogging.

Fix: Use 1–5 mL of overnight culture for standard spin columns. If the lysate is too thick, add more Buffer P1/P2/P3 to dilute it.

Genomic DNA (gDNA) contamination

The appearance of a high-molecular-weight "smear" or a distinct band above your plasmid on an agarose gel indicates host chromosomal DNA contamination.

Mechanical shearing

Unlike small circular plasmids, the large bacterial chromosome is extremely fragile. Vortexing or vigorous shaking after the addition of Buffer P2 (Lysis) or Buffer P3 (Neutralization) breaks the gDNA into small, linear fragments. These fragments then co-purify with the plasmid DNA on the silica membrane.

Fix: After adding Buffer P2 and P3, mix only by gentle inversion (6–8 times). Never use a vortex once the cells have been lysed.

Prolonged lysis

Exposure to the high-pH environment of Buffer P2 for minutes can permanently denature plasmid DNA, causing "shadow" bands that resist restriction digestion.

Fix: Move to the neutralization step immediately once the solution becomes clear and viscous. The total exposure to Buffer P2 should never exceed 5 minutes.

Culture overgrowth

Old or stationary-phase cultures contain a higher percentage of dead cells with pre-fragmented gDNA.

Fix: Harvest cultures at 12–16 hours. Avoid using cultures that have sat at room temperature or in the fridge for multiple days before processing.

RNA contamination

RNA carryover typically indicates that the RNase A enzyme—responsible for digesting host RNA—is missing or inactive.

Causes

• RNase not added to Buffer P1
• RNase degradation due to improper storage

Fix: Confirm RNase A addition and store Buffer P1 strictly at 4°C.

Low A260/A280 ratio (Protein contamination)

This reflects incomplete neutralization. If Buffer P3 is not mixed thoroughly, proteins will not precipitate efficiently.

Fix: Invert the tube immediately and thoroughly after adding Buffer P3 until the solution is uniform.

No DNA Band on Gel

If no plasmid appears, verify the following:

• Confirm antibiotic selection was maintained throughout the culture.
• Avoid over-drying the membrane during ethanol wash steps, as this can make DNA difficult to elute.
• Ensure elution buffer (preheated to 50°C for large plasmids) fully rehydrates the DNA on the membrane for 2 minutes before spinning.