Focus: DNA Purification

Remove the High-Speed Spin from PureYield™ Plasmid Preps

To eliminate the need for a high-speed centrifuge and to shorten processing times, we tested various matrices to remove bacterial cell debris by filtration rather than the high-speed centrifugation required by the PureYield™ Plasmid Midiprep and Maxiprep System protocols. Plasmid DNA purified from filtered lysates and centrifuged lysates performed similarly in restriction enzyme digestions and agarose gel electrophoresis, although DNA yield was lower with the filtration method.

By Rudy Zhao, Ph.D., Adam Petterson and Eric Vincent, Ph.D.
Promega Corporation

Published in October 2008

Introduction

The PureYield™ Plasmid Midiprep System (Cat.# A2492) provides a rapid method to purify 100–200μg of plasmid DNA from 50ml of bacteria culture. Plasmid DNA can be purified in as little as 30 minutes with the vacuum protocol, greatly reducing the time spent on purification compared to other DNA-binding matrix methods. The PureYield™ Plasmid Maxiprep System (Cat.# A2392) allows purification of up to 1mg of plasmid DNA from 250ml of bacterial culture in approximately 60 minutes. For best performance, the standard protocols for the PureYield™ Plasmid Midiprep and Maxiprep Systems require a high-speed centrifugation to clear cell debris before passing the lysates over the nested PureYield™ Clearing and Binding Columns (1). We tested various easy-to-get materials to filter the lysate and eliminate the need for the high-speed centrifugation.

Methods

E. coli JM109 containing moderate- (pGEM®-3Zf(+), Cat.# P2271) or high-copy-number plasmid (pGL3-Control Vector, Cat.# E1741) were cultured in LB medium supplemented with 100μg/ml ampicillin at 37°C overnight. Cells were pelleted by centrifugation at 5,000 × g for 10 minutes. Bacterial pellets were processed immediately or stored at –20°C for later use (fresh and frozen pellets gave comparable yields, data not shown). Plasmid was purified using the Vac-Man® Laboratory Vacuum Manifold (Cat.# A7231) and standard PureYield™ Plasmid Midiprep protocol (1) or PureYield™ Plasmid Maxiprep protocol (2) or using a protocol with a modified lysate-clearing step. For the PureYield™ systems, lysates were cleared by centrifugation at 15,000 × g for 15 minutes. In the modified protocol, this centrifugation step was replaced with filtration through one of the following materials: cheesecloth, gauze or coffee filter. In a single step, lysates were decanted through the indicated material and applied to the PureYield™ columns on the vacuum manifold. The Eluator™ Vacuum Elution Device (Cat.# A1071) was used to elute.

Results

PureYield™ Plasmid Midiprep System

The standard protocol for the PureYield™ Plasmid Midiprep System removes cell debris by centrifugation at 15,000 × g for 15 minutes before adding the cleared lysate to the PureYield™ Clearing and Binding Columns. We tested different filtration methods to remove cell debris from the neutralized cell lysate in place of the centrifugation step. Table 1 lists average DNA yield and purity.

Table 1. Average yield and purity of plasmids purified by different lysate-clearing methods. pGEM®-3Zf(+) Vector was purified from 50ml of overnight culture with the PureYield™ Plasmid Midiprep System and the indicated lysate-clearing method.
Lysate-Clearing Method Concentration (ng/μl) Yield (μg) A260/280 Ratio A260/230 Ratio
Standard protocol (centrifugation) 214 ± 18 104 ± 7.8 1.91 2.2
Coffee filter (one filter) 115 ± 39 45 ± 14 1.88 1.82
Cheesecloth (3 layers) 89 ± 10 35 ± 4.2 1.9 1.81
Gauze (3 layers) 122 ± 12 52 ± 4.1 1.88 1.88

The lysates cleared by filtration were visibly cloudy, unlike those processed using the standard centrifugation method. Washes took slightly longer with the filtration-cleared samples (up to 2 minutes versus less than 30 seconds). In addition, there was a 25–35% loss in lysate volume due to absorption and retention of lysate in the filtration materials; this led to a loss in yield (Table 1). The quality and digest performance of purified plasmids were indistinguishable by agarose gel electrophoresis (Figure 1).

thumbnail-BamHI digestion of plasmid DNA isolated using different lysate-clearing methods.
BamHI digestion of plasmid DNA isolated using different lysate-clearing methods

Figure 1. Digestion and agarose gel electrophoresis of plasmids purified using different lysate-clearing methods. Plasmid was isolated as described in the Methods section. The left panel shows uncut supercoiled plasmid; the right panel shows the same plasmid linearized by BamHI digestion. Lanes 1 and 5, plasmid purified from a fresh cell pellet using the standard method; lanes 2 and 6, plasmid purified from a frozen cell pellet using the standard method; lanes 3 and 7, plasmid purified from a lysate filtered through a coffee filter; lanes 4 and 8, plasmid purified from a lysate filtered through cheesecloth; lane M, BenchTop 1kb DNA Ladder (Cat.# G7541).

We focused on 3-inch gauze squares for filtration and tested 50ml and 100ml culture volumes with moderate- and high-copy-number plasmids (Table 2). Lysates from larger culture volumes were filtered easily through gauze (3 layers). Higher DNA concentration and purity were obtained with both plasmids at 100ml of culture.

Table 2. Average yield and purity of plasmids isolated from 50ml and 100ml cultures. Moderate-copy-number pGEM®-3Zf(+) Vector or high-copy-number pGL3-Control Vector was purified from 50ml or 100ml overnight cultures with the PureYield™ Plasmid Midiprep System using the standard method or gauze filtration for lysate clearing.
Lysate-Clearing Method Culture Volume Plasmid Concentration (ng/μl) Yield (μg) A260/280 Ratio A260/230 Ratio
Standard protocol (centrifugation) 50ml pGEM®-3Zf(+) 299 ± 23 103 ± 9.5 1.91 2.10
50ml pGL3-Control 873 ± 86 353 ± 18 1.91 2.24
Gauze filtration 50ml pGEM®-3Zf(+) 68 ± 14 23 ± 3.9 1.83 1.45
100ml pGEM®-3Zf(+) 186 ± 48 58 ± 17 1.94 1.92
50ml pGL3-Control 488 ± 42 160 ± 7.9 1.85 2.06
100ml pGL3-Control 1134 ± 54 287 ± 19 1.91 2.26

PureYield™ Plasmid Maxiprep System

We tested whether this filtration technique could be adapted to the PureYield™ Plasmid Maxiprep System, which requires the same high-speed centrifugation to clear lysates (2). For convenient handling of these high-volume lysates, we used a polypropylene funnel with a coffee filter insert. As with the Midiprep results, the filtered lysate was cloudy, solutions took longer to pass through these columns (up to 6 minutes versus less than 2 minutes) and yields were reduced (Table 3). The quality of plasmid purified using different methods was indistinguishable by agarose gel electrophoresis (Figure 2).

Table 3. Average yield and purity of plasmid purified with the PureYield™ Plasmid Maxiprep System. pGEM®-3Zf(+) Vector was purified from 250ml overnight cultures using the indicated lysate-clearing methods.
Lysate-Clearing Method Concentration (ng/μl) Yield (μg) A260/280 Ratio A260/230 Ratio
Standard protocol (centrifugation) 281 341 1.90 2.24
Coffee filter 150 180 1.89 1.95
 thumbnail-BamHI digestion of plasmid DNA isolated using the PureYield Maxiprep System.
BamHI digestion of plasmid DNA isolated using the PureYield Maxiprep System.

Figure 2. Example of plasmid purified with the PureYield™ Plasmid Maxiprep System and separated by agarose gel electrophoresis. The left panel shows uncut supercoiled plasmid; the right panel shows the same plasmid linearized by BamHI digestion. Lanes 1 and 3, plasmid purified from a fresh culture using the standard method; lanes 2 and 4, plasmid purified using the coffee filter method; lane M, BenchTop 1kb DNA Ladder (Cat.# G7541).

Summary

The high-speed centrifugation to clear debris from lysates in the PureYield™ Plasmid Midiprep and Maxiprep System protocols can be replaced by filtration through common gauze, cheesecloth or a coffee filter. This adds convenience, saves time and removes the need for a high-speed centrifuge. DNA yields are reduced, but the plasmid does not show additional nicking and performs well in restriction digestions. For highest DNA concentration and purity with the midipreps, we recommend using 100ml instead of 50ml cultures.

References

  1. PureYield™ Plasmid Midiprep System Technical Manual #TM253, Promega Corporation.
  2. PureYield™ Plasmid Maxiprep System Technical Manual #TM280, Promega Corporation.