Working with Plasmids
Troubleshooting the plasmid DNA purification
Step-by-step sample preparation for agarose gel analysis in plasmid purification
Save fractions from different steps of the purification procedure (see table Sample volumes required for agarose gel analysis). Remove aliquots from the cleared lysate (sample 1), flow-through (sample 2), combined wash fractions (sample 3), and eluate (sample 4), as indicated in each protocol and in the table below. Precipitate the nucleic acids with 1 volume of isopropanol, rinse the pellets with 70% ethanol, drain well, and resuspend in 10 µL TE buffer, pH 8.0. These aliquoted samples will be analyzed by agarose gel electrophoresis.
Sample volumes required for agarose gel analysis
Sample | Protocol step | Midi | Maxi | Mega | Giga | (Very low-copy plasmids/cosmids) QIAGEN-tip 100 |
(Very low-copy plasmids/cosmids) QIAGEN-tip 500 |
1 | 240 µL | 120 µL | 120 µL | 75 µL | 600 µL | 750 µL | |
2 | 240 µL | 120 µL | 120 µL | 75 µL | 50 µL | 24 µL | |
3 | 400 µL | 240 µL | 160 µL | 120 µL | 200 µL | 120 µL | |
4 | 100 µL | 60 µL | 22 µL | 20 µL | 50 µL | 30 µL | |
(% of prep represented by each sample volume) |
2% | 0.40% | 0.08% | 0.02% | 1% | 0.20% |
Analyzing plasmid DNA purification stages with agarose gel
L: Cleared lysate containing supercoiled and open circular plasmid DNA and degraded RNA (sample 1).
F: Flow-through fraction containing only degraded RNA is depleted of plasmid DNA which is bound to the QIAGEN resin (sample 2).
W: Wash fraction, in which the remaining traces of RNA are removed without affecting the binding of the DNA (sample 3).
E: The eluate containing pure plasmid DNA with no other contaminating nucleic acids (sample 4).
M: Lambda DNA digested with HindIII.
Lane 1: Supercoiled (lower band) and open circular form (upper band) of the high-copy plasmid pUC18 with an additional band of denatured supercoiled DNA migrating just below the supercoiled form. This form may result from prolonged alkaline lysis with Buffer P2 and is resistant to restriction digestion.
Lane 2: Multimeric forms of supercoiled plasmid DNA (pTZ19), which may be observed with some host strains, and should not be mistaken for genomic DNA. Multimeric plasmid DNA can easily be distinguished from genomic DNA by a simple restriction digestion — linearization of a plasmid sample displaying multimeric bands will yield a single defined band with the size of the linearized plasmid monomer (see lane 3).
Lane 3: Linearized form of plasmid pTZ19 after restriction digestion with EcoRI.
Lane 4: Sample contaminated with bacterial chromosomal DNA, which may be observed if the lysate is treated too vigorously (e.g., vortexing during incubation steps with Buffer P2 or P3). Genomic DNA contamination can easily be identified by digestion of the sample with EcoRI. A smear is observed, in contrast to the linear band seen after digestion of multimeric plasmid forms.
Lane 5: EcoRI digestion of a sample contaminated with bacterial genomic DNA which gives a smear above the plasmid DNA.
*When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs) from the product supplier.