Remove the High-Speed Spin from PureYield™ Plasmid Preps

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.

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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).

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).

Table 2.

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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.

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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.