100K device should be used to concentrate PCR products regardless of size if primer removal is required.
Materials of Construction
Nanosep Devices
Filter Media: Omega membrane (low protein-binding, modified polyethersulfone on polyethylene substrate)
Filtrate Receiver: Polypropylene
Nanosep MF Devices
Filter Media: Bio-Inert membrane (modified Nylon) or GH Polypro membrane (GHP, hydrophilic polypropylene)
Filtrate Receiver: Polypropylene
Effective Filtration Area
0.28 cm2
Dimensions
Overall Length (fully assembled with cap): 4.5 cm (1.8 in.)
Capacities
Maximum Sample Volume: 500 µL
Final Concentrate Volume: 15 µL
Filtrate Receiver Volume: 500 µL
Hold-up Volume (membrane/support): < 5 µL
Operating Temperature Range
0 - 40 °C (32 - 104 °F)
pH Range
Nanosep Devices: 1 - 14
Nanosep MF Devices: 3 - 14
Maximum Centrifugal Force
14,000 x g
Centrifuge
Fits rotors that accept 1.5 mL tubes
Sanitization
Provided non-sterile; may be sanitized by filtering 70% ethanol through the device prior to use
Choosing the Correct MWCO
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| MWCO |
Membrane Nominal Pore Size |
Biomolecule Size |
Biomolecule Molecular Weight |
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| 1K |
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3K - 10K |
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| 3K |
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10K - 20K |
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| 5K |
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15K - 30K |
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| 10K |
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30K - 90K |
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| 30K |
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90K - 180K |
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| 50K |
5 nm |
15 - 30 nm |
150K - 300K |
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| 100K |
10 nm |
30 - 90 nm |
300K - 900K |
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| 300K |
35 nm |
90 - 200 nm |
900K - 1,800K |
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DNA Recovery as a Function of Device MWCO
A 500 µL sample of a 100 µg/mL DNA fragment solution containing 50 and 500 bp double-stranded DNA fragments was centrifuged at 5,000 x g in Nanosep devices to a final volume of 50 µL. Recovered samples were quantitated using absorbance at 260 nm. The 100K device was able to differentiate between the sizes of the DNA fragments.
Omega™ Membrane has the Lowest Non-specific DNA Binding
A 500 µL sample of a 32P-labeled PCR* product (400 bp, 50 ng/mL) was centrifuged at 5,000 x g in a 100K Nanosep or competitive device. The retained DNA was recovered in 40 µL TE (10 mM Tris, 1 mM EDTA, pH 8). The remaining radioactivity in the device was counted in a scintillation counter. A value at 1000 CPM roughly corresponds to 1% of the total radioactive sample. Omega membrane has the lowest non-specific
binding, resulting in highest DNA recoveries. In general, DNA molecular weights do not correspond well with the MWCO because DNA is a long linear molecule. Effective retention of DNA by an ultrafiltration membrane requires a reduction in g-force to 5,000 x g. Otherwise, DNA can be forced through many MWCO membranes regardless of size.
*PCR technique is a proprietary technology of Hoffman-LaRoche.
DNA Recovery as a Function of Device MWCO
Nanosep 30K devices were used to filter dilute radioactive DNA fragments. In order to accurately quantitate DNA recovery from dilute samples, PCR products (100 and 400 bp) were dual labeled to low-specific activity with 32 P-labeled dCTP and 32P-labeled dATP and prepared for filtration. After synthesis, unincorporated nucleotides as well as termination products were removed by ultrafiltration using a 30K Nanosep device. The resulting retentate was checked for size and quantitated using gel electrophoresis. Labeled DNA in quantities ranging from 100 ng all the way down to 2 ng per device was diluted to 500 µL using TE. The samples (in triplicate) were centrifuged at 5,000 x g for 10 minutes (spun to dryness) and recovered in two washes of 20 µL water. The resulting retentate was added to a counting vial containing scintillation solution and counted.
Centrifugal Device Spin Times
Purification and Recovery of PCR Products
A small amount of 32P-labeled dCTP was added to the PCR reaction mix (Invitrogen) containing 32P-end labeled primers. The reaction was run for 30 cycles using standard conditions. Ten reactions were pooled and 100 µL aliquots were diluted to 500 µL and centrifuged in the indicated MWCO devices. The retained material was recovered in 20 µL TE, electrophoresed using a 10% polyacrylamide Tris Borate gel (BioRad) and analyzed by autoradiography. The 100K Nanosep device demonstrated the best combination of PCR product retention along with complete primer and nucleotide removal. If the desire is to ensure the removal of the buffer and free nucleotides but not primers, then the 30K or 10K devices will retain the PCR product while removing buffer components. Cleanup using the 10K device may be used if the PCR product is smaller than 200 bp and the presence of primers does not inhibit downstream applications
