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View additional product information for PureLink™ 96 Plasmid Purification System - FAQs (12263018)
22 product FAQs found
We typically recommend growing E. coli up to an optical density of 2.0 at 600 nm or a cell density of approximately 1 x 10^9 cells/mL in LB broth.
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.
1.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.
2.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).
3.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 see the detailed protocol below:
1.Equilibrate a column with 2 mL (mini)/10 mL (midi)/30 mL (maxi) of buffer EQ1.
2.Collect the phage lysate (liquid or plate lysis) and determine the exact volume.
3.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 degrees 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
4.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
5.To collect the phage particles, centrifuge for 10 minutes at greater than 10,000 x g. Discard the supernatant.
6.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
7.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 degrees C to lyse the phage particles.
X4 = 4% (w/v) SDS
8.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)
9.Wash the column with 2 x 2.5 mL (mini), 2 x 10 mL (midi), or 1 x 60 mL (maxi) of buffer W8.
10.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
11.Centrifuge the DNA for 30 minutes at ?13,000 x g at 4 degrees 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).
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.
Miniprep
Overnight bacterial culture volume: 1-3 mL
Approximate yield for high copy plasmids: ≤30 µg
Midiprep
Overnight bacterial culture volume: 15-25 mL
Approximate yield for high copy plasmids: 100-350 µg
Maxiprep
Overnight bacterial culture volume: 100-200 mL
Approximate yield for high copy plasmids: 500-850 µg
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.
For any silica columns, elution with water is generally possible. However, a buffer is preferred for stability and accuracy of absorbance readings, as pure water can have a very low pH (4 - 5).
Yes, the PureLink HiPure Plasmid kits can isolate BAC DNA, bacmid DNA, cosmid DNA, or M13 ssDNA. See the manual for a detailed protocol.
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, 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.
Endotoxins are typically any cell-associated bacterial toxins that are part of the outer surface of the cell wall of gram-negative bacteria. Endotoxins can influence cell growth, cell differentiation, contractility, and protein expression in mammalian cells. Endotoxins are released during bacterial lysis, and they can subsequently reduce transfection efficiency and protein expression levels. 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.
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.
The ratio of absorbance at 260 nm to the absorbance at 280 nm (A260/A280) is typically used to measure purity of the sample. For DNA, the ideal A260/A280 ratio is 1.8, but it can be in the range of 1.7 - 1.9. The A260/A230 ratio is also used to determine if contamination is present. For DNA, the ideal A260/A230 ratio is between 1.8 and 2.0. DNA purity 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). For genomic DNA, look for high average fragment sizes.
You are probably using too many cells.
Suggested solutions:
For Protocol A: Direct Load Method, cultures should be grown to OD600 of 4-8. If cultures are growing to a higher density, reduce incubation time in the future.
For Protocol B: Harvested Cell Method, use only LB medium. Cultures should be grown to OD600 of 1.8-2.8.
No. This product is not recommended for use with low-copy number plasmids when the plasmid DNA is for use in automated fluorescent DNA sequencing. Low-copy number plasmids can be used with this system when the samples are for use in PCR amplification. However, keep in mind that the yields from a low-copy plasmid may not be readily visible on a gel.
No. While the DNA is ideal for automated fluorescent sequencing, PCR, and restriction enzyme digestion, it is not compatible for eukaryotic transfection.
Plasmids up to 28 kb have been isolated and sequenced.
Yes. Keep the unused wells covered with Foil Tape. Please note that extra Foil Tape and Receiver Plates will be needed.
Generally speaking, yes. It should work the same if you can keep the same g (rcf) x time.
The original QiaRobot does not allow modifications to its protocol; however, more recent models are more flexible. Contact Technical Support to learn about robots that are compatible with the system.
For high-throughput quantitation, a method that uses PicoGreen or Hoechst Dye 33258 is recommended. Contact Thermo Fisher Scientific Technical Support for a protocol. The Quant-iT DNA Assay Kit, Broad Range is a fluorescence-based assay that can be used for accurate quantitation of DNA as well. A Lysis Buffer component makes A260/A280 ratios unreliable. However, the component does not affect DNA sequencing, PCR, or restriction enzyme digestion.