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Miniprep: Plasmid DNA Purification

Once you’ve picked and grown up an isolated colony, you now have a saturated culture containing billions of identical cells. Each cell carries multiple copies of the plasmid, and a miniprep allows you to extract and purify that plasmid DNA.

Why Miniprep?

  1. Storage — Purified plasmid can be stored at -20°C indefinitely.
  2. DNA as a Building Block — You often need clean DNA to build or clone further constructs.
  3. Sequencing — Verifying the plasmid sequence requires pure template.
  4. Transformation into Other Cells — Requires isolated DNA.
  5. General Use — Quantification, restriction mapping, and other analytical methods.

Overview

Qiagen QIAprep Miniprep Kit components Figure: Components of the Qiagen QIAprep Spin Miniprep Kit. Blue columns, labeled buffers, and RNase A tube shown.

This procedure is similar to the Zymo cleanup, but starts with a bacterial culture. We use the Qiagen QIAprep Spin Miniprep Kit, which uses silica column purification via guanidinium-silica chemistry. (This is not interchangeable with Qiagen anion-exchange kits.)

The protocol below is adapted from Qiagen's handbook, which includes detailed guidance, reagent preparation, and troubleshooting.
📄 Download the full handbook (PDF)

The Qiagen Miniprep Procedure

Illustrated summary of miniprep procedure showing steps from cell lysis to DNA elution. Step 1: Harvest and Lyse. Step 2: Clarify Lysate. Step 3: Bind Plasmid DNA. Step 4: Wash and Elute. Figure: Overview of the miniprep workflow — cells are lysed, debris is removed, DNA binds to a spin column, and is then washed and eluted.

🧪 Reagents

  • P1 Buffer (with RNase A)
  • P2 Buffer (NaOH/SDS)
  • N3 Buffer (Acidic, with guanidinium)
  • PB Buffer (protein/endotoxin removal)
  • PE Buffer (70% ethanol)
  • EB Buffer (elution)

🧫 Alkaline Lysis

  1. Pellet 1–5 mL saturated culture in a microcentrifuge tube.
  2. Resuspend in 250 µL P1 (RNase A must be added).
  3. Lyse by adding 250 µL P2, mix gently (do not vortex).
  4. Neutralize with 350 µL N3. Invert to mix thoroughly.
  5. Spin 5 min at max speed to pellet debris.

🧼 Column Binding and Wash

  1. Transfer supernatant to a blue QIAprep column, spin 15 s.
  2. Add 500 µL PB Buffer, spin.
  3. Add 750 µL PE Buffer, spin.
  4. Discard flowthrough, then spin again 90 s to dry.

💧 Elution

  1. Place column in a new 1.5 mL microcentifuge tube. Add 50 µL EB (or water, pH 7–8.5) to center of membrane.
  2. Spin 45 s to elute DNA.

Note: Avoid ethanol contamination from PE — spin thoroughly before eluting.

Lab Sheet Notes

  • Clearly label both the top and side of each tube with clone ID (e.g. pP6-79A).
  • Store the labeled miniprep tubes in the minis1 box.
  • Record miniprep location in the box (e.g., E3) on your lab sheet.
  • Minipreps are retained for future use and must be properly inventoried.

Kit Notes

  • Add RNase A to P1 before use; store in the fridge thereafter.
  • Add ethanol to PE buffer before first use. Waft the open tube to confirm ethanol has been added. A checkmark on the cap typically indicates ethanol has been added.
  • P2 buffer may precipitate when cold; ensure it is a clear liquid before use. Gently warm it (e.g., in the microwave) to fully dissolve any precipitate.
  • Buffers P2, N3, and PB contain irritants — wear gloves, goggles, and labcoat (as always).
  • Be mindful of contamination on your gloves. This procedure can be messy; crusty debris from bottle rims can transfer to gloves and contaminate your samples. Rinse your gloves at the sink if you suspect it.

🧪 Quiz: Miniprep

1️⃣ Elution Contents

What is in the tube after elution from the miniprep column?





2️⃣ Avoiding Errors

Which of these are good miniprep practices?