Home › Cell Biology › How to Perform Plasmid Prep
Steps
  1. 1 Pellet cells and resuspend in TE buffer 00:33
  2. 2 Lyse cells with denaturing solution 00:42
  3. 3 Renature and precipitate genomic DNA 00:56
  4. 4 Separate plasmid from precipitate 01:10
  5. 5 Precipitate and collect plasmid DNA 01:21
  6. 6 Wash and elute purified plasmid 01:33
  7. 7 Improve purity with optional treatments 01:47
  8. 8 Quantitate the purified plasmid DNA 02:10
Cell Biology Promega Corporation

How to Perform Plasmid Prep

Protocol
Difficulty
intermediate

Steps

1
Pellet cells and resuspend in TE buffer

Centrifuge the overnight bacterial culture to pellet the cells. Remove the supernatant and resuspend the cell pellet in TE buffer.

▶ 00:33
2
Lyse cells with denaturing solution

Add a denaturing solution to raise the pH and lyse the cells, releasing their contents into solution.

▶ 00:42
3
Renature and precipitate genomic DNA

Add renaturing solution to lower the pH, causing proteins and genomic DNA to precipitate while leaving plasmid DNA in suspension.

▶ 00:56
4
Separate plasmid from precipitate

Centrifuge the sample to pellet proteins and genomic DNA. Transfer the supernatant containing plasmid DNA to a new tube.

▶ 01:10
5
Precipitate and collect plasmid DNA

Add ethanol to precipitate the plasmid DNA, then either centrifuge to pellet it or transfer the sample to a DNA-binding column.

▶ 01:21
6
Wash and elute purified plasmid

Wash the pellet or column with 70% ethanol to remove salts and contaminants. Elute or resuspend the DNA using water or neutral buffer.

▶ 01:33
7
Improve purity with optional treatments

Optionally enhance plasmid purity by adding RNase A after lysis or performing a phenol-chloroform extraction to remove remaining contaminants.

▶ 01:47
8
Quantitate the purified plasmid DNA

Measure the concentration and purity of the final plasmid DNA solution before proceeding to downstream applications or assays.

▶ 02:10

🚨 Failure Case Library (3) + Submit your own case

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
severe
Partial Digest with Multiple Uncut Bands
Gel shows mixture of fully digested, partially digested, and uncut DNA. Some recognition sites cleaved while others remain intact in the same molecule.
💡 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
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