Plasmid DNA Purification Support—Getting Started
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We offer three main technologies for plasmid purification: anion exchange, silica, and ChargeSwitch® chemistry.
The ratio of absorbance at 260 to the absorbance at 280 nm (A260/A280) is typically used to measure purity of the sample. For DNA, the ideal ratio is 1.8, but could be in the range 1.7–1.9. The A260/A230 ratio is also used to determine if contamination is present. The ideal ratio for DNA is 1.8–2.0. Purity of DNA can also be examined by gel analysis. For plasmid DNA, look for a strong, single band (perhaps with a few extra bands representing multimers of the desired molecule).
Anion exchange purification is recommended for higher purity and lower endotoxin levels. Silica-based purification is not optimal for transfection, as there is a higher level of endotoxins and impurities. Larger plasmids also work better with anion exchange columns.
Endotoxins are typically any cell-associated bacterial toxins that are part of the outer membrane of the cell wall of gram-negative bacteria. Endotoxins can influence cell growth, cell differentiation, contractility, and protein expression in mammalian cells. During bacterial lysis, endotoxins are released, and can reduce transfection efficiency and subsequently protein expression levels. Plasmid DNA purified with our PureLink® HiPure plasmid kits has endotoxin levels that are equivalent to endotoxin-free kits (0.1 EU/µg) for superior downstream performance. Please review the following article for more information about endotoxins: Butash KA et al. (2000) Reexamination of the effect of endotoxin on cell proliferation and transfection efficiency. Biotechniques 29(3): 610-614, 616, 618-619.
Yes, we offer our EveryPrep™ Universal Vacuum Manifold (Cat. No. K2111-01), which allows for direct elution from the manifold using our ChargeSwitch® Pro Filter Plasmid Mini, Midi, and Maxi Kits.
Column-based Plasmid Purification
All kits termed “HiPure” use anion exchange resin columns to isolate the highest quality plasmid DNA, suitable for transfection. Filter kits include a filter to clear bacterial lysate without centrifugation, while the HiPure FP kits include a filter and precipitator to eliminate the need for centrifugation at either the bacterial lysate clearing or DNA precipitation step.
Yes, the PureLink® HiPure Plasmid kits can isolate BAC DNA, bacmid DNA, cosmid DNA, or M13 ssDNA. See the manual for a detailed protocol.
We would recommend using our PureLink® Quick Plasmid Kits. These preps are silica based, with fast protocols (approximately a 45 minute centrifugation). While the isolated plasmid DNA is lower in purity than DNA isolated using our PureLink® HiPure kits, it is suitable for sequencing and cloning.
HQ stands for “higher quality” silica, which provides slightly higher purity plasmid DNA than the PureLink® Quick Plasmid Kits. Endotoxin units are <40 EU/µg using the HQ silica. The plasmid DNA isolated using the PureLink® HQ kits is suitable for cloning, sequencing, and some transfection protocols.
For any silica columns, elution with water is generally possible. A buffer is preferred for stability and accuracy of absorbance readings, as pure water can have very low pH (4–5).
We do not recommend decreasing the volume of elution buffer, as this will cause yield to drop. You can try to perform an additional elution to increase yield.
Please see the chart below for our typical yields:
Overnight bacterial culture volume
Approximate yield for high copy plasmids
Up to 30 µg
Up to 150 µg
Up to 750 µg
Up to 2.5 µg
Up to 10 mg
The PureLink® HiPure Mini, Midi, and Maxi Kits can purify plasmids up to 200 kb, while the PureLink® HiPure Mega and Giga Kits can purify plasmids up to 150 kB.
Yes, please see the detailed protocol below:
- Equilibrate a column with 2 mL (mini)/10 mL (midi)/30 mL (maxi) of buffer EQ1.
- Collect the phage lysate (liquid or plate lysis) and determine the exact volume.
- According to the scale, add 30 μL/100 μL/400 μL of buffer X1 to 10 mL/50 mL/250 mL phage lysate and incubate at 37°C for 30 min.
X1 = 100 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10 mM EDTA, 20 mg/mL RNase A, 6 mg/mL DNase I
- Mix the nuclease digest from step 3 with 2 mL/ 10 mL/50 mL ice-cold buffer X2 and incubate on ice for 60 min.
X2 = 3 M NaCl, 30% (w/v) polyethyleneglycol (PEG) 6000
- To collect the phage particles, centrifuge for 10 minutes at >10,000 x g. Discard the supernatant.
- Resuspend the pelleted phage particles in 1 mL (mini)/3 mL (midi)/9 mL (maxi) buffer X3 with a pipet.
X3 = 100 mM Tris-HCl (pH 8.0), 25 mM EDTA
- Add 1 mL (mini)/3 mL (midi)/9 mL (maxi) of buffer X4 to the phage suspension. Mix thoroughly by inverting the tube several times and incubate for 10 minutes (mini) or 20 minutes (midi and maxi) at 70°C to lyse the phage particles.
X4 = 4% (w/v) SDS
- Add 1 mL (mini)/3 mL (midi)/9 mL (maxi) of buffer X5 to the lysate, mix thoroughly by inverting, and centrifuge for 10 minutes at room temperature and ≥13,000 x g. Collect the supernatant without taking too many particles and apply it directly onto the equilibrated column (see step 1). Allow the lysate to enter the resin by gravity flow.
X5 = 3.0 M potassium acetate (pH 5.5 with acetic acid)
- Wash the column with 2 x 2.5 mL (mini), 2 x 10 mL (midi), or 1 x 60 mL (maxi) of buffer W8.
- Elute the lambda DNA from the column with 0.9 mL (mini)/5 mL (midi)/15 mL (maxi) of buffer X6 and precipitate the DNA by adding 0.7 volumes of isopropanol, previously equilibrated to room temperature.
X6 = 100 mM sodium acetate (pH 5.0 with acetic acid), 1,500 mM NaCl
- Centrifuge the DNA for 30 minutes at ≥13,000 x g at 4°C. Because lambda DNA is very sticky, it will spread over the whole wall of the centrifuge tube if a fixed angle rotor is used. Therefore, we recommending the use of a swinging bucket rotor (i.e. HB-4 or HB-6 for Sorvall centrifuges), or, if such a rotor is not available, using centrifuge tubes (i.e., Corex) siliconized with dimethyldichlorosilane.
After centrifugation, was the lambda DNA with 80% ethanol and dry it briefly. Dissolve the lambda DNA in a suitable amount of TE or 10 mM Tris buffer (pH 8.0).
- Lysis conditions vary greatly for gram-positive bacteria, so you would need to start with a specific lysis protocol that is known to work for the particular bacteria you are working with. The lysis materials and conditions in the kit may not work well.
- After lysis using the specific protocol, precipitate the crude plasmid DNA with either 70% ethanol or isopropanol. Be sure that the genomic DNA is removed (follow precautions in the package insert; do not vortex at any time).
- Resuspend the resulting nucleic acid pellet in a small volume (1 mL for mini, 10 mL for midi, 24 mL for maxi) of 600 mM NaCl, 100 mM sodium acetate, pH 5.0.
Apply this solution to an equilibrated column and continue with the standard protocol.
Yes, please follow our suggested protocol:
- 1) Lyse mammalian cells with a common mammalian lysis procedure, for instance, alkaline lysis. Please note, be careful to be gentle in this step, as sheared genomic DNA will copurify with plasmid.
- 2) Precipitate DNA with ethanol or 70–80% isopropanol. This is a crude precipitate.
- 3) Resuspend in 600 mM NaCl, 100 mM NaOAC, pH adjusted to 5.0.
- 4) Load onto equilibrated HiPure column.
This should work for plasmids up to 100 kb. Note that mitochondrial DNA will also copurify.
We typically recommend growing E. coli up to 1–1.5 A600 units/mL (~1 x 109 cells/mL) in LB broth.
Magnetic Bead-based Plasmid Purification
ChargeSwitch® Technology features a charged nucleic acid binding surface that is “switchable” by changing the pH of the surrounding buffer. At low pH, the surface is positively charged and binds the negatively charged nucleic acid backbone, allowing easy removal of proteins and other contaminants using a simple wash step.
The typical yield is 25 µg from a 5 mL culture for the ChargeSwitch® Pro mini kit, 100–200 µg from a 25 mL culture for the midi kit, and 500–1,000 µg from a 100 mL culture from the maxi kit. The plasmid size range for the kits is typically 3–9 kb.
The filter kit is based upon a dual nested column design, which facilitates the fastest and simplest protocol for obtaining high quality plasmid (<10 EU/µg DNA). Modified wash buffer 1 removes more endotoxin.
Automated Plasmid Purification
Yes, we offer the BenchPro® 2100 Plasmid Purification System, which is a fully automated maxiprep purification system.
While yield and purity will differ depending on the size of your vector and copy number as well as cell strain used, a high copy vector (pcDNA3.1) has been tested in several popular strains including C600, DH10B/T1R, DH5alpha, JM109, TOP10, and XL1-Blu, demonstrating an average yield above 500 µg.
MC5001 is lightweight and has a small footprint. This compressor includes a drying unit, which is a dual moisture trap and membrane dryer. The noise level is 60 dB. This works well in a common lab area where –80°C freezers and fume hoods are kept. The MC6001 is our ultra-quiet, oil-filled compressor. It is heavier and taller than MC5001. It also comes with the same dryer unit to reduce moisture.
For LB medium, you can fill the reservoir to the maximum volume of 150 mL. For TB medium, though we have not tested this, we have heard from customers that a 50 mL culture diluted in 50 mL medium works well.
You can pipette out 700 µL of TE, so that the final DNA concentration could be higher. We would not recommend using less than 750 µL TE buffer.
A self test is performed each time a run is started and takes approximately 6 minutes to complete.
You can use a Hedland air gauge to measure the air flow rate. The minimal specification is 3 cubic feet per minutes. There is no upper limit for flow rate.
You will need an in-house air supply outlet (80–100 psi, 3 cfm, constant flow rate), or a compressor unit with the same specifications, that can be connected to the BenchPro® 2100 Plasmid Processing Station.
No, the instrument works using positive air pressure, and cannot work on vacuum. It works using house air, which is the orange line that forces air out. The pressure has to be 80–100 psi.
You can run one or two samples simultaneously on the BenchPro® 2100 Plasmid Processing Station.
No, the BenchPro® 2100 Plasmid Purification Card is not reusable. During the purification, E. coli cells are captured and lysed within the card, making subsequent reuse impossible.
The waste tray is reusable and is made of material that can be autoclaved. You can also order new waste trays.
No. The cell liner tray and lid are not reusable. They should be disposed of after each procedure. Each BenchPro® 2100 reagent kit comes with a supply of cell liner trays and lids. Cell liner trays are not currently available for standalone purchase.
Always store the BenchPro® 2100 Plasmid Card and BenchPro® 2100 Reagent Tray in the supplied box to prevent any damage to the card sippers or to the tray foil. Store both at room temperature.
Yes, you can use an air compressor. Please contact Technical Support for recommended models. The air compressor will need to:
- Regulate pressure output between 80 and 100 psi
- Regulate pressure output flow at >3 cfm, when measured at 80 psi
- Regulate output flow to <5°C dewpoint
The BenchPro® 2100 instrument needs a manual setup time of less than 5 minutes. After setup, the instrument takes about 90 minutes for the entire purification process. You do not need to be present during the purification process.
Yes. If you wish to obtain a higher concentration of plasmid DNA, you can do so by reducing the volume of elution buffer in the corresponding compartment of the reagent tray. To reduce the volume of elution buffer, use the pipette tip to pierce the foil, then draw out a corresponding volume of buffer. For specific instructions on how to do this, please contact Technical Service.
The instrument is capable of purifying plasmids from 2.7 to 20 kb.
Yes. Resuspend the cell pellets in 100 mL of LB medium before you load the mixture onto the cell liner tray.
On average, you can process 0.7 to 0.9 g of wet pellet biomass on this instrument. However, this is subject to variability when working with cells of different size and different strains.
The purified plasmid is ultrapure and suitable for all downstream applications, including those requiring the highest purity, such as transfection of mammalian cells, automated and manual DNA sequencing, PCR amplification, in vitro transcription, bacterial cell transformation, cloning, and labeling.
You can clean the surface of the BenchPro® 2100 instrument with a damp cloth. Do not use harsh reagents or solvents to clean the unit. Clean the bottom of the instrument drawer with a light spray of 70% ethanol and then wipe off the ethanol with a paper towel.
For best results, follow these guidelines:
- Use a bacterial culture at a cell density of approximately 109 cells/mL, or an optical density of 2.0 to 2.4 at 600 nm (OD600). Use bacterial culture in transition between exponential and stationary phase.
- Use a high copy number plasmid to obtain a good yield of plasmid DNA. High copy number plasmids typically yield 2–6 µg DNA/mL LB culture grown overnight.
We do not recommend the use of TB or rich media. A high cell density will clog the membrane and decrease DNA yield from the purification. For best results, set up the purification as indicated in the manual.
Yes. Water can be used to replace the 1.5 mL TE buffer for elution. Carefully pierce only the TE-containing chamber and remove all TE. Rinse the chamber twice with purified water and then fill with 1.5 mL purified water.
We have tested DH10B-T1R, DH5α, JM109, TOP10, and XL1-Blue in-house, and expect that any strains derived from these will also work. Note: endA+ strains have been shown to work, but there may be nuclease activity in the final purified plasmid.
No. Purification of BAC clones is not optimal due to their large size. The BenchPro®2100 instrument is capable of purifying plasmids from 2.7 to 20 kb.
Yes. You can resuspend the pellet in LB media or sterile water such that the OD at 600 nm is 2.0.
By simply using a USB device, you can download any available software updates.
After downloading the software updates, insert your USB device in the slot on the back of the instrument, and then follow instructions on the screen.
No, the current version of the BenchPro® 2100 cannot be connected to any laboratory information management systems.
No, the current version of your software will not allow you to interrupt and subsequently continue your run.
For Research Use Only. Not for use in diagnostic procedures.