Lysate Clearance for Prokaryotic DNA Isolation Using the AcroPrep 96 Filtration Plate

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By Debi Morris and Kevin Seeley

1. Introduction

Use of Filtration for Lysate Sample Preparation

Molecular biology methods have led to the successful production of a variety of biomolecules from prokaryotic and eukaryotic cell lines. Synthesized biomolecules include DNA, RNA and proteins but the most common biomolecule produced is in the form of plasmid DNA. Prokaryotic cells are widely used to produce recombinant DNA molecules for research applications ranging from in vitro protein synthesis to cosmid library screens. The scale or volume of the sample is dependant on the downstream applications. Currently, the large-scale purification schemes of the past have given way to the development of small-scale massively parallel purifications requiring automated/ semi-automated handling.

The most problematic step in the purification of plasmid DNA is the clarification of the sample once the cells are lysed and the lysate has been treated with salts to precipitate the detergent. The lysate often contains biomolecule concentrations that could be millions of times higher than the molecule of interest in a soup contaminated with a large load of cellular debris. The clarification step is necessary to remove the cellular debris. In the past, centrifugation was the primary method used to sediment the loose pellet.

Sedimentation of cellular debris has several limitations that present roadblocks to many applications that require small-scale high throughput processing. It is very difficult to sediment the fluffy pellet in a multi-well plate while still recovering supernatant. Other problems with sedimentation include long spin times, rotor capacity for single tube processing and handling issues associated with using microfuge tubes. Perhaps the greatest problem is that sedimentation allows the fluffy pellets to trap a large portion of the desired sample since pellets are notoriously difficult to ‘wash’ without fragmenting the plasmid samples.

In contrast, filtration can be automated, is relatively quick and allows for use of an additional wash step to maximize sample recovery. Filtration can be done effectively in either a vacuum or centrifugal mode, ultimately maximizing the choice in protocols available to the researcher. We report the comparison of filtration to sedimentation as well as the added benefit of using a plate configuration that allows the incorporation of prefiltration.

Filter Plates

The proprietary design of the AcroPrep 96-well plate configuration incorporates the ability to insert a variety of microporous and ultrafiltration membranes. It is designed for low-biomolecule binding, low weeping and high chemical resistance. Most critical, however, is the ability to seal multiple layers of membranes into the device using the proprietary compression seal. This feature is what allows the plate to have a prefilter over a more restrictive microporous membrane, combining the benefits of fast flow rates for particulate-laden solutions with the ability to clarify the sample. Other plate schemes would require two plates to achieve this level of clarity.

2. Procedure

Lysis Procedure

We describe the standard lysis procedure but recognize that many others exist (Maniatis et al.).

1. Grow E. coli JM 109 containing plasmid vector pCAT 3-control to log phase.

2. Decant 1.5 ml of the overnight culture into microfuge tubes.

3. Centrifuge for one minute to pellet cells.

4. Remove supernatant by aspiration.

5. Re-suspend the cell pellet in 100 µl 10mM EDTA 25mM Tris-HCl pH 8.0 containing 50 µg/ml Rnase.

6. Store for five minutes at room temperature.

7. Add 200 µl freshly prepared 0.2M NaOH 1% SDS and mix gently by inversion.

8. Store on ice for five minutes.

9. Add 150 µl ice-cold potassium acetate (pH ~4.8).

10. Transfer flocculent lysate to an AcroPrep 96 BioInert filtration plate with prefilter.

11. Proceed to vacuum or centrifugal filtration.

Vacuum Filtration

Place filter plate containing a prefilter along with a chemically compatible receiver plate on the vacuum manifold.

1. Apply vacuum. Most house vacuum sources do not exceed 15 inches Hg (38.1 cm Hg); however, AcroPrep96 is capable of tolerating much higher vacuum pressures.

2. Release vacuum slowly. (Do not release vacuum by pulling the corner of the plate, because it will degrade the manifold gasket.)

3. Discard filter plate and disassemble vacuum manifold to retrieve receiver plate.

4. Transfer the filtrate on to an appropriate plate for downstream applications.

Centrifugal Filtration

1. Place filter plate containing a prefilter on top of a compatible receiver plate.

2. Insert filter and receiver plates together into a standard swinging bucket microtiter plate rotor assembly. A multiple stack with the GHP plate over the 10K plate on top of a sturdy receiver may also be an option.

3. Centrifuge. As a general guideline, centrifugation at 500 xg for one to two minutes is sufficient to evacuate contents of well. Centrifugation times will vary depending on solution viscosity and type of membrane.

4. Transfer the filtrate on to an appropriate plate for downstream applications.

3. Results and Discussion

The use of filtration for the clarification of plasmid purification lysates enables a rapid and effective alternative to centrifugal sedimentation. The use of a prefilter shortens filtration times (Table 1) and allows greater flow rates to be maintained at higher loads . In addition, the prefiltration process allowed the highest A260 recovery with ratios equivalent to sedimentation (Fig. 2). Typically A260/A280 ratios of 1.9 indicate a more pure DNA sample, supporting the benefit of prefiltration for effective clarification. Agarose gel electrophoresis of the samples confirms that the amount of DNA recovered, shown by absorbance values, was highest for the BioInert plus prefilter plate (0.32 units) (Fig. 3). The gel band intensities correlate well with the fact that the sample is richer in plasmid DNA using the AcroPrep 96 BioInert with a prefilter.

Debi Morris and Kevin Seeley, PhD, Scientific and Laboratory Services, Pall Corp.