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Plasmid Minipreparation using MultiScreen 96-Well Filter Plates

Introduction

The need for rapid, cost effective high throughput methods for plasmid preparation has been steadily increasing. This technical note details a protocol enabling the use of MultiScreen filtration plates and user-supplied reagents to complete plasmid purification in the 96-well format without the need for expensive kits. The resultant purified plasmid DNA is of high quality and fully functional as assayed by cycle sequencing with dye terminators. Sequencing accuracy of greater than 99% at 600 bases is routinely achieved on the APB MegaBACE 1000 (see Figure), PE Biosystems ABI 3700, and slab gel DNA sequencers.

The protocol was developed and optimized based on standard methods. This rapid and automation-friendly protocol clears the lysate by filtration and relies primarily on vacuum for sample processing, delivering 3-4 g of plasmid per well from two deep 96-well blocks within 60 min. The processing time required for more than two plates depends on the number of available vacuum manifolds.

The method is automation compatible. Protocols are available for the Beckman Biomek® 2000, Packard MultiPROBE II, and the Robbins Hydra 96® liquid handlers.

Other mini-prep protocols utilizing MultiScreen plates are described in the literature.^{1, 2}

Protocol ³

1. Inoculate E. coli into 1 ml aliquots of 2X LB⁴ plus appropriate antibiotic in sterile 96 deep well blocks (2 ml capacity). Cover plates and secure in incubator. Incubate at 37C at 320 r.p.m. for 20 to 24 hours (The optical density at 650 nm should be approximately 1.35).
2. Cover the deep well block cultures with clear plate tape (Millipore: MATA 096 00), and centrifuge at 1500 x g for 5 min (or alternatively, 1000 x g for 10 min). After centrifugation, immediately decant culture supernatant to a container for proper disposal. Invert and tap the plates firmly on several layers of paper towels on the bench to remove residual culture supernatant.
3. Resuspend pellets in 80 L of Solution I first using a plate shaker or vortex, and then mix by pipetting. [Complete resuspension is important].
4. Add 80 L of Solution II. Mix immediately and vigorously with a plate shaker (maximum speed) for 1 min. Incubate for an additional 2-min. at room temperature. [Do not expose lysate to Solution II for more than 5 min or plasmids may denature irreversibly].
5. Add 80 L of Solution III. Mix immediately and vigorously (maximum speed) with a plate shaker for 2 min.
6. Separately, add 150 L of Bind Solution to each well of a MultiScreen FB plate (Millipore: MAFB N0B 50). Place the FB plate in the bottom of the vacuum manifold (Millipore: MAVM 096 0R, or equivalent).
7. To remove the lysate, lower the pipette tips down the sides of the deep wells through the lysates until reaching bottom. Pipette up and down three times. Slowly remove 180 L. of lysate from the bottom of each deep well, and dispense into the corresponding well of a MultiScreen-NA lysate clearing plate (Millipore: MANANLY50). Entering the same wells a second time, remove any residual lysate from the deep well plate, and transfer it to the corresponding wells of the MultiScreen lysate clearing plate. [The second transfer attempt will be almost entirely air, but is included in case any of the tips failed to fill on the first try. The important thing is to transfer the whole lysate. Considerable cell debris and bubbles may be visible in the pipette tips, but this is not a cause for concern.]
8. Place the NA plate on top of the manifold, and adjust the vacuum to 8 inches of Hg (0.27 bar - 203 torr.) Do not exceed 8 inches Hg vacuum setting during filtration of the lysate to ensure uniform filtration). Apply the vacuum for 3 min, drawing the lysate through the NA plate into the FB plate prefilled with Bind Solution from step 6. Discard NA plate.
9. Remove the FB plate from inside the manifold and mix the cleared lysate thoroughly with Bind Solution by rapidly pipetting up and down several times.
10. Place the FB plate on top of the empty manifold. Apply full vacuum for 1 min. Direct filtrate to waste. Plasmid DNA is now bound to the FB plate.
11. Add 200 L of 80% ethanol (reagent grade is adequate) to each well of the FB plate. Apply full vacuum for 1 min. Direct filtrate to waste.
12. Repeat step 11, but apply vacuum for 3 min.
13. Remove the FB plate from the manifold. Blot the bottom of the plate on a clean, lint-free absorbent material.
14. Place the FB plate on top of a standard microtiter plate with centrifuge alignment frames (Millipore: MACF09604) and centrifuge at 1000 x g for 10 minutes to dry.
15. To dissolve plasmid, add 70µL of TE buffer to each well of the FB plate. This protocol consistently delivers 3-4 g of pLH2 plasmid DNA (at 70-90 ng/l) from E. coli JM109 as measured fluorometrically (Hoechst 33528). Yields will vary depending on replicon and insert. If higher concentrations (100-150 ng/L) are required, dissolve in 50 L of TE instead of 70 L in step 15. The smaller elution volume will deliver plasmid which on average will be approximately 50% more concentrated, but the average total yield will be 20% lower.. Deliver TE close to the center of each well.
16. To elute plasmid, centrifuge at 1000 x g for 5 min (eluate volume is typically 45 µL), or, elute by vacuum.

Solution Preparation

Solution I (30 mM Glucose; 15 mM Tris-HCl, pH 8; 30 mM Na2EDTA; 60 g/ml RNase A)

41.45 ml Milli-Q water
1.5 ml filter-sterilized 1 M Glucose
6 ml 250 mM Na2EDTA
0.75 ml 1 M Tris-HCl
0.3 ml 10 mg/ml RNase A
50 ml = Vf -Store in refrigerator.


Solution II (0.2 N NaOH; 1% SDS)

17.6 ml Milli-Q water
0.4 ml 10 N sodium hydroxide
2 ml 10% SDS
20 ml = Vf -Seal tightly, and store at room temperature.


Solution III (3.6 M Potassium; 6 M Acetate)

14 ml Milli-Q water
14 ml concentrated glacial acetic acid
72 ml 5 M potassium acetate
100 ml = Vf -Store at room temperature.


Bind Solution (6.1 M KI)

40 g potassium iodide
28 ml Milli-Q water
40 ml = Vf -Store at room temperature in brown bottle wrapped in aluminum foil.



2X LB (double strength LB, except NaCl)

20 g tryptone
10 g yeast extract
10 g sodium chloride
1000 ml Milli-Q water
-Autoclave 100 ml aliquots. Store at room temperature. Add antibiotic just before use.


TE Buffer (10 mM Tris-HCl, pH 8; 1 mM Na₂EDTA)

1 ml 1 M Tris-HCl, pH 8
0.4 ml 250 mM Na₂EDTA
98.6 ml Milli-Q water
100 ml = Vf -Autoclave, and then store at room temperature.

¹Engelstein et al. 1998. Microbial & Comparative Genomics. Volume 3, Number 4. ,

²Itoh et al. 1997. Nucleic Acids Research. 25:1315-1316

³This method is used at the Sanger Center (http://www.sanger.ac.uk/Teams/Team51/Vacprep2.shtml), and at other key facilities involved in the Human Genome Project.

⁴The choice of culture medium impacts the overall performance of the protocol. Normal strength LB results in low plasmid yield. Very rich culture media such as Terrific Broth interferes with filtration of the cleared lysate. Inoculation and incubation of 1 ml of double-strength LB (all components are 2X normal, except NaCl which is 1X) for 20 hours at 320 rpm in deep well plates ensures that the cultures are saturated (O.D. 650 nm ~1.35). Shorter incubation times can lead to well-to-well variability of cell density which in turn can negatively effect the consistency of plasmid yields. An antibiotic concentration of 100 g/ml is recommended for multicopy vectors requiring ampicillin. Under these conditions, plating efficiencies are identical under selective and non-selective conditions, suggesting there is no plasmid loss. Before incubation, cover your deep well plates so that they remain axenic, but allow for aeration (i.e., not airtight). We suggest breathable plate tape such as Diversified Biotech breath-easy gas permeable sealing membrane for microtiter plates, cat. No. BEM-2X, 100 sheets/pack.

⁵The choice of culture medium impacts the overall performance of the protocol. Normal strength LB results in low plasmid yield. Very rich culture media such as Terrific Broth interferes with filtration of the cleared lysate. Inoculation and incubation of 1 ml of double-strength LB (all components are 2X normal, except NaCl which is 1X) for 20 hours at 320 rpm in deep well plates ensures that the cultures are saturated (O.D. 650 nm ~1.35). Shorter incubation times can lead to well-to-well variability of cell density which in turn can negatively effect the consistency of plasmid yields. An antibiotic concentration of 100 g/ml is recommended for multicopy vectors requiring ampicillin. Under these conditions, plating efficiencies are identical under selective and non-selective conditions, suggesting there is no plasmid loss. Before incubation, cover your deep well plates so that they remain axenic, but allow for aeration (i.e., not airtight). We suggest breathable plate tape such as Diversified Biotech breath-easy gas permeable sealing membrane for microtiter plates, cat. No. BEM-2X, 100 sheets/pack.

⁶ This protocol consistently delivers 3-4 g of pLH2 plasmid DNA (at 70-90 ng/l) from E. coli JM109 as measured fluorometrically (Hoechst 33528). Yields will vary depending on replicon and insert. If higher concentrations (100-150 ng/l) are required, dissolve in 50 l of TE instead of 70 l in step 15. The smaller elution volume will deliver plasmid which on average will be approximately 50% more concentrated, but the average total yield will be 20% lower.


Ordering Information

Product Name	Device Color	Device Materials	Sterility	Qty/Pk	Membrane	Catalog Number
MultiScreen-FB	opaque	Barex/TiO2	non-sterile	10	1.0 µm FB glass fiber	MAFBN0B10
MultiScreen-FB	opaque	Barex/TiO2	non-sterile	50	1.0 µm FB glass fiber	MAFBN0B50
MultiScreen-NA	clear	Styrene	non-sterile	50	NA	MANANLY50
Vacuum manifold basic kit, includes manifold base, standard ring with gaskets, support grid, all tubing, valves and pressure gauge				1		MAVM0960R