Protocol: Plasmid DNA Purification using the QIAprep Spin Miniprep Kit and a Vacuum Manifold

critical

Confirmed colony later shows recombination or deletion

Initial screening looked correct, but on re-verification the insert is missing pieces or the vector has rearranged.

💡 4 · ✓ 4

critical

No DNA Recovered from Plasmid Miniprep

No detectable plasmid DNA is recovered after miniprep purification. Column elution yields no nucleic acid by spectrophotometry or gel electrophoresis.

💡 4 · ✓ 4

critical

No DNA Recovered from Gel Extraction or PCR Cleanup

No DNA is recovered after gel extraction or PCR cleanup procedures. Elution yields no measurable nucleic acid.

💡 3 · ✓ 3

severe

Double digest band pattern does not match prediction

Picked colonies grow normally but the diagnostic double-digest yields bands of unexpected size on the gel.

💡 5 · ✓ 5

severe

Low Plasmid Yield Due to Incomplete Lysis

Plasmid miniprep yields are significantly below expected levels. A260/280 ratio may be normal but total DNA concentration is low.

💡 5 · ✓ 5

severe

Mini-prep yields almost no plasmid DNA

Eluate DNA concentration is very low; diagnostic digest and gel show no detectable target band.

💡 4 · ✓ 4

severe

Low Plasmid Quality Due to Degradation or Denaturation

Plasmid DNA shows smearing on gel, poor A260/280 ratio, or fails in downstream applications despite adequate concentration. Multiple bands or reduced supercoiled form may be visible.

💡 5 · ✓ 5

moderate

Co-transfection ratio imbalance — uneven dual-plasmid expression

Of two co-transfected plasmids, only one expresses strongly; the fraction of double-positive cells is low.

💡 5 · ✓ 4

moderate

Low DNA Yield from Gel Extraction or PCR Cleanup Due to Incomplete Elution

DNA recovery from gel extraction or PCR cleanup is lower than expected based on input amount. Some DNA likely remains bound to column.

💡 4 · ✓ 4

moderate

Low DNA Performance in Downstream Applications Due to Contaminants

Purified DNA fails or performs poorly in downstream applications (transformation, restriction digestion, PCR, sequencing) despite acceptable concentration and A260/280 ratio.

💡 6 · ✓ 6