Plasmid Prep Kits

Use endotoxin-free (EF) kits when preparing plasmid DNA for transfection of mammalian cells, especially sensitive primary cells or for in vivo applications. Endotoxin (LPS) contamination can activate immune responses and reduce transfection efficiency. Standard minipreps are fine for sequencing, restriction digestion, and cloning.

Typical requirements: 0.5–2 µg per well in a 6-well plate, 5–10 µg for a 10 cm dish, and 20–50 µg for electroporation or large-scale transfection. A single midiprep (100–200 µg) provides enough DNA for most cell culture transfection experiments.

Yes. Miniprep DNA purified on silica columns is suitable for Sanger sequencing. Most sequencing facilities require 100–200 ng/µL in 10–15 µL, which is easily achieved from a standard miniprep. Ensure the A260/280 ratio is above 1.8 for clean sequencing reads.

Common causes: insufficient culture growth (harvest at OD600 ≥1.5), low-copy-number plasmid (expected), incomplete lysis (ensure thorough but gentle mixing), or overloaded column (do not exceed the recommended culture volume). Also verify that the resuspension buffer contains RNase A.

Store eluted plasmid DNA in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) or nuclease-free water at -20°C for long-term storage. For frequent use, aliquot and store at 4°C for up to 1 month. Avoid repeated freeze-thaw cycles. EDTA chelates divalent cations, protecting DNA from nuclease degradation.