Working with Plasmids

Troubleshooting the plasmid DNA purification

Thi: Hi Dr. Davis, I’m having some trouble with the quality of plasmid that I’m getting after plasmid purification. I’m not sure what I might be doing wrong and was hoping if you could help me troubleshoot?

Dr. Davis: Of course! I’d be happy to help. Several different factors can cause poor plasmid DNA yields and quality. Let’s discuss how to determine at what stage of the procedure the problem occurred.

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

Following the agarose gel electrophoresis protocol, run 2 μl of each sample on a 1% agarose gel to analyze the fractions of each stage of the plasmid purification procedure. The figure titled “Agarose gel analysis of the plasmid purification procedure” illustrates an analytical gel of the fractions, together with examples of the problems that can arise at each step. If you find that you have a problem with a particular step of the protocol, turn to the hints in the relevant section of the troubleshooting guide in the handbooks. If the problem remains unresolved, or if you have any further questions, please call QIAGEN Technical Service.

Agarose gel analysis of the plasmid purification procedure.

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.