Lysate Clearance for Prokaryotic DNA Isolation Using the AcroPrep™ 96 Filter Plate

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. A 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 create roadblocks to many applications where small-scale, high-throughput processing is required. First, it is very difficult to sediment the fluffy pellet in a multi-well plate and recover the 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 plasmid DNA. (Pellets are notoriously difficult to wash without fragmenting the plasmid samples.)

Filtration, which is easily automated, is relatively quick and allows the 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 an integral prefilter for lysate sample preparation.

AcroPrep 96 Filter Plates

The proprietary design of the AcroPrep 96 filter plate enables the incorporation of a variety of microporous and ultrafiltration membranes. It is designed for low biomolecule binding, low weeping, and high chemical resistance. Most critical is the ability to use the proprietary sealing technology to seal multiple layers of membranes into the filter plate. This feature allows the AcroPrep 96 filter plate to have a larger pore size prefilter over a more restrictive microporous filter polishing, combining the benefits of fast flow rates for particulate-laden solutions with the ability to clarify the sample. Competitive plate schemes require two plates to achieve this level of clarity.

AcroPrep 96 filter plates have been designed to:

• Reduce crosstalk – proprietary sealing technology individually seals membrane in each well, reducing well-to-well crosstalk.
• Add versatility – available in 96- and 384-well formats. The product portfolio includes a variety of membranes and configurations, plate colors, well volumes, and outlet tip lengths for use in a multitude of sample preparation and detection processes.
• Assure consistency – designed in accordance with the standards of the Society for Biomolecular Screening (SBS). The single-piece design strengthens these plates for automated applications.
• Provide confidence – the chemically resistant/biologically inert polypropylene housing construction is low protein and nucleic acid binding, and durable when used with harsh chemicals.
• Make it convenient – serialized barcode allows the use of automated tracking systems.
  

1. Grow E.Coli JM 109 containing plasmid vector pCAT 3 to log phase.
2. Decant 1.5 mL of the overnight culture into microfuge tubes.
3. Centrifuge for 1 minute to pellet cells.
4. Remove supernatant by aspiration.
5. Resuspend the cell pellet in 100 µL 10mM EDTA 25mM Tris-HCl pH 8.0 containing 50 µg/mL RNase.
6. Store for 5 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 5 minutes.
9. Add 150 µL ice-cold potassium acetate (pH ~4.8).
10. Transfer flocculent lysate to an AcroPrep 96 filter plate with Glass Fiber over Bio-Inert® membrane (PN 5046).
11. Proceed to vacuum or centrifugal filtration.

Vacuum Filtration

Place the AcroPrep 96 filter plate (PN 5046), along with a compatible receiver plate, on a vacuum manifold.

1. Apply vacuum to filter the lysate. Most house vacuum sources do not exceed 15 in. Hg (38.1 cm Hg); however, the AcroPrep 96 filter plate is capable of tolerating vacuum pressures as high as 29 in. Hg (73.7 cm Hg).
2. Release vacuum slowly. (Do not release vacuum by pulling the corner of the plate as it will degrade the manifold gasket.)
3. Discard the AcroPrep 96 filter plate (PN 5046) and disassemble vacuum manifold to retrieve receiver plate.
4. Filtrate is now ready for downstream applications. 

Centrifugal Filtration

1. Place the AcroPrep 96 filter plate (PN 5046) on top of a compatible receiver plate.
2. Stack filter and receiver plates together and place into a standard swinging bucket microtiter plate rotor assembly.
3. Centrifuge. As a general guideline, centrifugation at 500 x g for 1 to 2 minutes is sufficient to evacuate the well contents.
4. Filtrate is now ready for downstream applications.
   

The AcroPrep 96 filter plate with an integral prefilter configuration improves the time and effectiveness of filtration by allowing the prefiltration of viscous samples. Once clarified, the sample can be desalted using an AcroPrep 96 ultrafiltration plate (see Technical Protocol, “Desalting/Buffer Exchange for Biomolecules Using AcroPrep 96 UF Filter Plates,” PN 33309). These results are possible because of the proprietary AcroPrep 96 filter plate construction, which is unique in its ability to allow multiple layers to be independently sealed into each well.

Figure 1
Flow Rate Comparisons at Two Sample Volumes

Filtration times were measured for 100 µL and 200 µL lysate samples. Average flow rates were calculated for the AcroPrep 96 filter plate with Bio-Inert membrane (PN 5042), AcroPrep 96 filter plate with Glass Fiber over Bio-Inert membrane (PN 5046), and a Competitor hydrophilic plate containing a floating prefilter (COMP). Error bars indicate standard error (n=8).

Table 1
Vacuum Filtration Times at Three Sample Volumes

0.2 µm Bio-Inert Membrane (PN 5042)

3.0 µm Glass Fiber/Prefilter over 0.2 µm Bio-Inert Membrane (PN 5046)

Competitor*

*An equivalent competitor 96-well plate with a single hydrophilic microporous membrane with a floating prefilter. Pore size is not indicated. Filtration times were measured for 100, 200 and 300 µL lysate samples (n = 5). Vacuum was applied at 25.4 inches Hg (64.5 cm Hg). All tests were performed using the Porvair (Shepperton, UK) manifold. Typical filtration times are presented in seconds.

Figure 2
Clarification Effectiveness as a Measure of Absorbance

The UV absorbance of the supernatant or filtrate was measured using a UV spectrophotometer at 260 and 280 nm. The plot shows the ratios of A260 over A280 readings for AcroPrepTM 96 filter plate with Bio-Inert membrane (PN 5042), AcroPrep 96 filter plate with Glass Fiber over Bio-Inert membrane (PN 5046), Competitor plate (COMP), and Sedimentation (SED). Pall’s prefiltration plate had the highest recovery of DNA and a ratio closest to sedimentation.

Figure 3
Plasmid Recoveries Shown by Gel Electrophoresis

Agarose gel electrophoresis of the sedimented or filtered samples. A = AcroPrep 96 filter plate with Bio-Inert membrane (A260 of 0.28), B = AcroPrep 96 filter plate with Glass Fiber over Bio-Inert membrane (A260 of 0.32), C = Competitor plate (A260 of 0.15), and D = sedimentation (A260 of 0.11). HinDIII/lambda molecular weight markers were used at each end of replicate samples. The far-left sample in each panel is a purified plasmid control. Gel-mobility-shift for the replicate samples is due to the high salt in the lysate. Note: Effective desalting can be achieved using AcroPrep 96 ultrafiltration plates (see Technical Protocol, “Desalting/Buffer Exchange for Biomolecules Using AcroPrep 96 UF Filter Plates,” PN 33309).

General Notes on Multi-well Filtration
Solution Weeping
Weeping is defined as the seepage or spontaneous dripping of fluid through the membrane. Weeping leads to loss of solution/sample from the wells, resulting in reduced signal detection and/or sample recovery. Weeping is also messy and can cause contamination and problems with automated instrumentation. The AcroPrep 96 filter plate is engineered to minimize weeping, permitting fluid flow only when force (e.g., vacuum, centrifugation) is applied.

Crosstalk
Crosstalk refers to situations where the content of one well “leaks” into adjacent wells. Crosstalk can occur upstream between wells in a plate or downstream following filtration into a receiver plate. Elimination of crosstalk upstream in the filter plate using the AcroPrep 96 filter plate is assured through innovative design and sealing features such as individually cut, placed, and sealed membranes. For applications that require filtrate collection, crosstalk can occur downstream in the receiver plate. This potential problem has been dramatically reduced in the AcroPrep 96 filter plate by engineering outlet frits (flow directors) optimized for minimal sputtering during filtration, and splash guards in the event that sputtering does occur.

• Product Data, AcroPrep Membrane-bottom Filter Plates, PN 33296 (384 wells, 1 mL, 350 µL)
• Technical Report, Automated Purification of Combinatorial Libraries Using AcroPrep 96 Filter Plate with GHP Membrane, PN 33245
• Technical Protocol, Desalting/Buffer Exchange for Biomolecules Using AcroPrep 96 UF Filter Plates, PN 33309
• Brochure, Discover Endless Potential, Products for Genomics, Proteomics, and Drug Discovery, PN 33286

• Centrifugal Devices for nucleic acid and protein sample preparation in the following sample volumes:

• Nanosep MF (microfiltration) Centrifugal Devices are available with Bio-Inert or GHP membranes for low protein binding and high recoveries in applications such as particulate removal prior to sample analysis (GHP product is HPLC grade) and removal of precipitates.
• BioTrace™, Biodyne®, and Fluorotrans® Transfer Membranes offer precise performance and compatibility with nearly every detection system available.
• AcroWell™ and AcroPrep 96 and 384-well Filter Plates are an excellent platform for a wide variety of molecular biology, analytical, and high throughput sample preparation and detection applications.