Scientific & Technical Report PN 33205

Single-tube DNA Purification and Using Ultrafiltration Devices

Authors: Fang Qi1, Kevin Seeley2, and Timothy W. Short1

1 Biology Department, Queens College and the Graduate School of the City University of New York, 65-30 Kissena Boulevard, New York, NY 11367-1597 2 Pall Life Sciences, Scientific and Laboratory Services, 600 South Wagner Road, Ann Arbor, MI 48103-9019

Abstract The resulting ligation-ready DNA fragments were Traditional methods for subcloning DNA fragments ligated to digested vector directly within the or shuttling sequences between vectors involve ultrafiltration device, and then transformed into the ligation of fragments from competent cells. Using Pall Life Sciences Nanosep chromosomes, , cosmids, or PCR products devices, we were able to achieve significant savings into specific sites on a selected . In in time, labor, and starting materials, while the order to desalt, remove primers or adapters, or resulting yield of transformed recombinants concentrate DNA fragments, researchers generally roughly equaled that of the traditional techniques. use and precipitation steps that are labor-intensive and time-consuming. In Introduction an effort to develop a more streamlined process, Standard techniques require we compared traditional methods of subcloning enzymatic modification and purification steps to PCR-generated fragments against a rapid procedure prepare DNA sequences for ligation and using Nanosep® centrifugal ultrafiltration devices. transformation into bacterial cells. In the past, After optimizing the parameters for effective target procedural steps have included either serial or DNA purification, we synthesized PCR products simultaneous double restriction digests followed with terminal adapter sequences containing by gel electrophoresis to purify away adapter restriction sites. These adapters were digested, sequences. If not removed, the remaining adapter generating the appropriate cohesive ends for ligation. sequences compete for ligation sites and The resulting DNA products were concentrated significantly increase the number of spurious and desalted while simultaneously allowing the recombinants and reduce recombination primers and adapter fragments to pass into the efficiency. Because of the multiple purification discarded filtrate during a five-minute centrifugation steps with low recovery yields obtained with step (Figure 1). standard techniques, these methods require large amounts of starting DNA to guarantee sufficient Figure 1 vector and insert material to complete subsequent Directional Subcloning of PCR Products ligation and transformation steps (1). Synthesize PCR with Primers Containing Restriction Digest Sites Standard subcloning protocols incorporate a -based positive selection such as ampicillin resistance, allowing researchers to identify which Site A Site B colonies are transformed with vector sequences. Vector components such as the LacZ gene Cut and Remove Adapter A encoding the b-galactosidase enzyme can be A used to determine which antibiotic-resistant Cut and Remove Adapter B transformants are recombinant by blue/white A B screening in the presence of the X-gal substrate. Ligate and Transform Prepared Insert If an insert interrupts the polylinker in the LacZ, gene expression is disrupted and the ampicillin- resistant transformant will appear white. Typically, the first step in the transfer of a DNA sequence for A pool of PCR products was divided into two identical cloning involves digestion of the DNA by restriction enzymes aliquots. One fraction was precipitated with 1/10 volume 3 M to produce terminal sequence overhangs that facilitate ligation sodium acetate and 2 volumes ethanol, chilled at -20 °C for into a similarly prepared vector. Following each digestion step, 2 hr, centrifuged at high speed (12,000 x g) for 30 min, rinsed any compatible adapter sequences need to be removed so they with 70% ethanol, and air dried prior to resuspension in 40 µL will not compete with the desired insert during subsequent sterile distilled H2O (sdH2O). The second fraction was diluted ligation. The most frequently used method for this purification to 500 µL and centrifuged at 5,000 x g for 15 min in a is to separate adapters from the desired DNA electrophoretically, Nanosep 30K device. The retained material was resuspended and then to recover the DNA from the gel. Because of the in 40 µL dH2O and divided into 10 µL aliquots for analysis. numerous applications that require production of recombinant One aliquot was used as an uncut control while the other DNA molecules, a rapid method is needed for transferring aliquots were diluted into a 20 µL restriction digestion reaction. DNA fragments from a PCR product or other source into the These samples were digested with EcoRI, BamHI, or XbaI desired vector. (Promega, Madison, WI, USA) for 30 min at 37 °C, electrophoresed on a 1.5% agarose gel, and the DNA was Materials and Methods visualized under UV light after staining with ethidium bromide. Optimized DNA Separation and Recovery Rapid Cloning of PCR Fragments Using Ultrafiltration To test the utility of Nanosep devices in practical cloning ® To test the ability of Nanosep ultrafiltration devices to applications, we generated a PCR product from the fern discriminate between PCR products and primers, the recovery Ceratopteris richardii corresponding to a 620 bp conserved characteristics of DNA molecules of two different sizes were region of a phytochrome (PHY) photoreceptor genomic DNA compared for devices containing different MWCO (molecular sequence (3). Genomic template DNA from three 14-day-old weight cutoff) membranes. A 500 µL sample at 100 µg/mL C. richardii gametophyte tissue was isolated by the alkaline (50 µg total) DNA containing either a 50 bp or 500 bp lysis method (7). Forward (5'-AAGGATCCACACGGGAAGTTT- double-stranded DNA fragment was centrifuged at 5,000 x g TGGCC-3') and reverse (5'-TTAAGCTTGATATTCGGGACTCT- in Nanosep devices and recovered in 50 µL TE. Recovered GAAAC-3') primers were constructed with sequences samples were quantitated spectrophotometrically using corresponding to complementary sequences from a genomic absorbance at 260 nm. PHY gene. adapter sites for BamHI and The effectiveness of ultrafiltration for PCR product purification HinDIII were engineered two base-pairs downstream from the was examined by observing the recovery of free nucleotides, 5' ends of each primer, respectively, to generate compatible primers, and PCR products following filtration though a selection sticky ends for cloning into the vector polylinker (underlined in of devices containing different MWCO membranes. Before the above sequences). The 25 µL PCR reactions were starting PCR, γ-32P-dATP end-labeled primers as well as 1 µL prepared using PCR Master Mix (Qiagen, Valencia, CA, USA) α-32P-dCTP were added to the PCR reactions. The 400 bp supplemented to 2.5 mM MgCl2, 0.2 µM primers, and 1 µL DNA fragments were synthesized using a custom primer set genomic template DNA. PCR reactions were transferred to a (5'-ACCTATAGCGGTGTGAAATACCGCACAGATGCGTAAG- preheated thermocycler at 90 °C and incubated for 1 min, GAGA-3' and 5'-TGGGCGCTCTTCCGCTTCCTCGCTCACT- then subjected to 35 cycles of 30 sec at 94 °C, 30 sec at GACTCGCTGCGCT-3') corresponding to sequences 45 °C, and 60 sec at 72 °C, followed by a postcycle extension approximately 200 bp upstream and 200 bp downstream for 3 min at 72 °C. A 6 µL aliquot of each reaction was of the pUC18 polylinker. All radioactive PCR products were subjected to eletrophoresis to confirm the size and purity of synthesized at low specific activity by adding 1 µL of labeled the products and to quantitate the fragments for subsequent nucleotide (α-32P-dCTP, 3000 Ci/mmol) to the PCR reaction. cloning. The utility of Nanosep device-based cloning was After synthesis, unincorporated nucleotides were removed by compared with standard fragment cloning methods (5). ultrafiltration (Nanosep 30K device at 5,000 x g for 5 min). The Nanosep device method: To minimize handling time and labor, resulting retentate was recovered in 40 µL TE (10 mM Tris, 1 µg of PCR product and 0.2 µg of pBluescript KS+ vector 1 mM EDTA, pH 7.5) and quantified using gel electrophoresis. (Strategene, La Jolla, CA, USA) were combined and The reaction was run for 30 cycles using standard conditions; simultaneously digested with BamHI and HinDIII directly in a the samples were pooled to ensure consistency, then split into Nanosep 100K device. Digestion was carried out in a total equal aliquots for desalting and concentration in a variety of volume of 50 µL for 2 hr at 37 °C. The digestion products MWCO devices. The retained material was recovered in 20 µL were centrifuged through the Nanosep device at 1,500 x g for TE, electrophoresed using a 10% polyacrylimide Tris-borate gel 5 min, and the retentate was washed twice by addition of 50 µL (BioRad, Hercules, CA, USA), and analyzed by autoradiography. dH2O and centrifugation at 1,500 x g for 5 min. The retained DNA was then resuspended in 8.5 µL sdH2O, 1 µL 10x ligation The retention of biological activity during DNA handling is buffer with ATP, and 0.5 µL ligase. Ligation was allowed to critical. To determine the effect of ultrafiltration on biological proceed directly in the Nanosep device at 17 °C overnight. activity, we synthesized a 400 bp PCR product containing a centrally located polylinker (described above), purified the Standard method: Classic methods were applied for cloning product by ultrafiltration or ethanol precipitation, and tested the PCR product into a cloning vector, and the results were its suitability for digestion. Analysis of the restriction digest compared with those obtained by the Nanosep device-based fragments of this PCR product allowed us to determine method. The PCR product was purified away from primers whether the DNA could be recovered without interfering and free nucleotides by gel electrophoresis on low melting with subsequent enzymatic manipulations. point agarose. The appropriate gel band was excised and the DNA recovered by melting the agarose at 60 °C and diluting Figure 2 the material to 500 µL with dH2O. The DNA was purified by Size Separation of DNA Using Ultrafiltration for Primer Removal first partitioning the sample into 500 µL 25:24:1 phenol: chloroform:isoamyl alcohol, then repartitioning the resulting (A) 100% 50 bp aqueous phase into 500 µL chloroform and precipitating the 500 bp DNA from the aqueous phase with 50 µL of 3 M sodium 90% acetate and 1 µL ethanol. The DNA was chilled for 2 hr at 80% -20 °C, pelleted by centrifugation at 12,000 x g for 30 min, 70% and washed with 500 µL 70% ethanol (5). The precipitated 60% DNA was air-dried and resuspended in 10 µL dH2O, and 1 µL was subjected to gel electrophoresis for quantitation. The 50% cleaned PCR product (5 µg) and a separate aliquot of 40% pBluescript KS+ (2 µg) were digested with BamHI and HinDIII. 30% After the DNA was restricted for 2 hr at 37 °C, the digestion Recovery Percent products were separated electrophoretically, the appropriate 20% bands were excised from the gels, and the DNA was recovered 10% from the low melt agarose and extracted as described above. 0% The PCR product (1,000 ng) and vector (200 ng) were 3K 10K 30K 100K combined and ligated in a total volume of 12 µL as described MWCO Device for the Nanosep® device method. Transformation and Recombinant Analysis A 3 µL aliquot of ligation product from either the Nanosep C 3K 10K 30K 100K 300K device or standard method was used to transform competent (B) XL1-Blue cells (Strategene, La Jolla, CA, USA). Cells from each transformation were plated onto LB plates with ampicillin 400 bp to select for transformants and X-gal and IPTG for blue/white colony selection (5). Transformation efficiencies and white:blue colony ratios were calculated for each preparation, and several Primers white colonies from each were analyzed by PCR for insertion of the phytochrome fragment. White colonies from each Nucleotides preparation were transferred to a fresh LB + ampicillin plate and allowed to grow overnight at 37 °C. A swipe of each (A) Ultrafiltration-based separation of DNA fragments by size. DNA fragments colony was picked with a sterile toothpick and resuspended in (50 mg) were recovered in a variety of Nanosep devices. The recovered DNA 7.0 µL H2O. After lysing the cells for 10 min at 98 °C, a PCR was quantitated using absorbance at 260 nm. Error bars indicate the standard error of three independent experiments. (B) Separation of target DNA from mixture containing Qiagen Master Mix, 2.5 mM MgCl2, and primers and free nucleotides by ultrafiltration. Radioactive nucleotides, 2 µM primers was added; the PCR reactions were then cycled primers, and PCR products were recovered in a variety of Nanosep devices. according to the same procedure used to amplify the original Sample fractions were electrophoresed in a 10% TBE polyacrylamide gel. Equivalent band intensities approximate 100% recovery of the 400 bp PCR phytochrome gene sequences. PCR products were subjected product, the 22 base primers, and the unincorporated nucleotides. to agarose gel electrophoresis to assay the proportion of desired recombinants obtained by the standard and the Separation of Primers and Adapters Nanosep device method. Based on the differential separation seen in Figure 2A, an experiment was devised to verify which ultrafiltration devices Results would be most useful for the removal of primers and free Separation and Recovery of DNA Fragments Using nucleotides from PCR reactions. End-labeled primers together Ultrafiltration with radioactive nucleotides were added to a PCR reaction Previous empirical data from the manufacturer’s application and a 400-bp product was synthesized using standard PCR guide (6) indicate that the Nanosep ultrafiltration devices, methods. The resulting solution contained labeled DNA prod- which were originally optimized for protein recovery, could also ucts as well as the primers and unincorporated nucleotides. be used to concentrate and desalt DNA fragments. In addition Consistent with data in Figure 2A, the Nanosep 3K device to desalting, the Nanosep devices are capable of separating two only removed a fraction of the free nucleotides while the mixed DNA fragments as long as their sizes are significantly Nanosep 10K and 30K devices effectively removed free different. When either a 50 or 500 bp fragment is purified nucleotides but not the primers (Figure 2B). At the same time, using a variety of MWCO devices, the 3K, 10K, and 30K the Nanosep 300K device allowed all of the DNA and precur- devices retain both species of DNA. In contrast, the 100K sors to pass into the filtrate. Finally, the Nanosep 100K device device is able to retain the larger fragment and allow the was able to differentially separate the PCR products from the smaller fragment to pass into the filtrate (Figure 2). This obser- mixture of primers and nucleotides in a single spin. In addition vation indicates that the Nanosep 100K ultrafiltration device to differential separation, the retained material in the 100K can be used effectively for the removal of primers, polylinkers, device was concentrated, desalted, and ready for down- and adapters from mixed DNA fragment solutions. stream enzyme modifications.

www.pall.com/lab It is critical that the act of concentration and PCR product to BamHI digestion steps followed by additional ultrafiltration purification using ultrafiltration does not somehow interfere do not result in a significant loss of sample, based on the with the downstream modifications required for further digestion observed recoveries (Figure 4, lanes 3 through 6). Similar and ligation. To determine whether ultrafiltration damages or results were obtained for serial digestions performed in the contaminates DNA samples, experiments to test the ability of BamHI to HinDIII direction and for simultaneous digestion with the DNA to be cleaved by a variety of restriction enzymes both enzymes. Serial digestion of the cloning vector was also were performed. Pooled, unlabeled PCR fragment samples performed, and the small excised polylinker was efficiently were purified using either ultrafiltration or ethanol precipitation, removed from the ligation-ready vector by ultrafiltration in a and then each was split into four samples. One sample Nanosep 100K device (not shown). served as a control while the others were digested with three different restriction enzymes that were selected for their Figure 4 sensitivity to sub-optimal conditions. The data indicate that Effect of Terminal Restriction Digestion on Subsequent all three enzymes cut the ultrafiltration-prepared target DNA to Ultrafiltration Retention completion, as evidenced by the presence of a 200 bp doublet rather than an uncut 400 bp fragment (Figure 3). In contrast, PCR HinDIII BamHI PCR samples purified by ethanol precipitation did not digest to completion when using the more demanding XbaI enzyme, (-) (+) (-) (+) (-) (+) suggesting that residual salts or ethanol had inhibited the reaction. Figure 3 Digestion Efficiency of DNA Purified by Ultrafiltration or Ethanol Precipitation

Pooled PCR Reaction

30K Nanosep® Device Precipitate A radioactive, dilute (100 ng) PCR product amplified from a cloned fragment C E B X C E B X of Ceratopteris phytochrome DNA template was purified using a Nanosep 100K device. The restriction digest products are shown for the first (HinDIII) and second (BamHI) restriction digests. (-) = sample prior to Nanosep device concentration; (+) = recovered concentrated sample after filtration. Equivalent 400 bp band intensities approximate 100% recovery. 200 bp Rapid Preparation of DNA Fragments The ultimate test of the effectiveness of the Nanosep device Ultrafiltered or ethanol precipitated PCR products (400 bp with a pUC19 method is to prepare vector and insert DNA by ultrafiltration, polylinker in the center) were digested using a variety of restriction enzymes. and then to ligate and transform the products into cells. The A doublet at approximately 200 bp indicates the appropriate digestion of the 400 bp fragment. C = Uncut Control sample, E = Cut with EcoRI, B = Cut recovery of recombinants obtained by this method can then be with BamHI, and X = Cut with XbaI. compared with that observed using DNA purified by standard gel-based methods. Electrophoretic methods are able to PCR techniques allow researchers to amplify a DNA sequence separate completely the primer and adapter sequences, but from a target template mixture. To facilitate molecular cloning, they require subsequent purification of the desired DNA 5'-terminal sequences containing restriction enzyme recognition fragment from the gel prior to use. By contrast, ultrafiltration sites (adapters) can be included during primer synthesis. This methods have no electrophoresis steps, although trace strategy aids subsequent ligation of a restricted PCR fragment amounts of primers, adapters, and polylinkers may be into an appropriately digested and purified vector. Because of retained in the membrane, potentially interfering with the their small size, adapter fragments can be removed by following cloning steps. Therefore, it was important to ultrafiltration following restriction digestion while simultaneously determine whether these trace contaminants would significantly concentrating the sample and allowing buffer exchange. hinder recombination of the desired fragment. PCR-fragment subcloning has often been problematic partly The preparation of vector and PCR products using standard due to the ineffective digestion of adapters as well as the low methods involved the separate digestion and purification of recombinant yields obtained with traditional techniques. The both vector and insert sequences. The use of ultrafiltration for ability of ultrafiltration to facilitate the molecular cloning of a PCR DNA sample preparation allowed two variations of the product was demonstrated using a serial restriction digest ultrafiltration protocol to be performed. In the first method performed on a phytochrome gene fragment. The fragment was (UF Method A), the vector and insert DNA molecules were amplified from Ceratopteris richardii genomic DNA template digested and purified separately, and mixed only at the ligation using gene-specific primers containing short terminal HinDIII step. In the second method (UF Method B), the vector and and BamHI adapters. The removal of free nucleotides was insert DNA molecules were mixed together at the start of the shown for the PCR sample purification [Figure 4, left two restriction digestion, allowing them to be digested, purified, lanes, before (-) and after (+) ultrafiltration]. Subsequent HinDIII and ligated in a single Nanosep 100K device. Regardless of which method was used to generate Figure 6 recombinants, the number of recombinants recovered was PCR Products Resulting From the Amplification of DNA roughly the same for three independent experiments (Figure Templates From Individual White Colonies 5). The largest difference was in the time and labor involved performing the procedure. Gel electrophoresis purification PCR Verification of Subclones steps that normally require 2 to 3 hr to perform could be replaced by a simple 5 min centrifugation step. The rapid ® ® Nanosep method using Nanosep device-based ultrafiltration gave Device more non-recombinant colonies than the standard method, (Rapid) consistent with the fact that trace polylinker sequences were likely to be present; but the use of the LacZ marker system effectively alleviates this shortcoming. In addition, using a third or fourth enzyme that cuts the polylinker, but not the insert, could help remove most of the non-recombinant clones (8). Standard Figure 5

Comparison of the Number of Recombinant Colonies for The 620 bp band visualized on an agarose gel indicates successful Independent Experiments, Each Using Three Different Sample recombination of the amplified gene fragment into the vector. The Preparation Procedures absence of a 620-bp product indicates a spurious recombination event.

200 Gel Method Discussion 175 UF Method A The availability of selective and differential culture media UF Method B allows the use of rapid molecular cloning techniques. The 150 ability to purify and digest DNA vectors and inserts using 125 ultrafiltration not only allows rapid subcloning with smaller

100 initial quantities of DNA, but it helps to circumvent some of the other obstacles frequently encountered during standard 75 gel purification-based methods. Among the most serious of Recombinants 50 the problems of traditional techniques are carry-over of salts and residual ethanol or other organic solvents that can inhibit 25 downstream enzymatic reactions. Furthermore, the yields for 0 DNA recovery from gels and precipitation are often low, 123 requiring large amounts of starting sample and extensive Independent Experiment labor. In this report we demonstrate that ultrafiltration with White colonies from a standard transformation protocol using equal volumes Nanosep devices is well suited for the high-yield, rapid of ligation product from each method were counted. Gel Method = insert and recovery of dilute DNA samples without the retention of vector DNA were prepared using gel purification techniques as described in the text; UF Method A = insert and vector DNA were prepared in separate components that may inhibit biological activity. We present tubes using the rapid Nanosep device methods; UF Method B = insert and a method for cloning PCR products or other DNA fragments vector DNA were combined and prepared together using a single Nanosep with significant advantages of time, labor, and materials over 100K device. classical methods, and with comparable yields of desired recombinants. Although ultrafiltration has been shown Recombinant insert analysis was done by performing colony previously to be an efficient method to purify nucleic acids PCR with the same primer set used to generate the insert. (2, 4), to our knowledge this is the first report showing the Colonies were considered positive for the insert if the PCR efficacy of ultrafiltration in practical cloning procedures. produced a band of the appropriate size. Putative This report shows that the ultrafiltration method has distinct, recombinant colonies (white) were screened and, for the quantifiable advantages over other commonly-used subcloning standard method, 17 out of 19 colonies (89%) contained the techniques. The speed and efficiency of this method, along correct insert. When the rapid method transformants were with the availability of Nanosep devices with different MWCO screened, 14 out of 17 colonies (82%) contained the correct membranes, suggest this basic method could be adapted to insert (Figure 6). The slightly higher number of spurious cloning strategies for DNA of different sizes from many sources. recombinants for the rapid method is likely due to the presence of trace adapter sequences. While it is possible Acknowledgements that some enzyme activity could be retained during The authors thank Ms. Jeanette Kew-Chao for providing ultrafiltration (unless a phenol:chloroform step is used or genomic DNA and for technical support. This work was heating to 80 °C for 10 min) it is apparent from these data supported in part by funds from Pall Life Sciences. that any carry-over of enzymes did not interfere with the recovery of recombinants. The fidelity of subcloning was comparable to that of traditional methods and therefore is sufficient for routine subcloning procedures.

www.pall.com/lab References 1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1996. Current Protocols in . John Wiley & Sons, New York. 2. Henderson, M. B., and A. M. Krowczynska. 1992. Efficient purification of PCR products using ultrafiltration. BioTechniques. 13:286-289. 3. Kolukisaoglu, H. U., S. Marx, C. Wiegmann, S. Hanelt, and H. A. W. Schneider-Poetsch. 1995. Divergence of the phytochrome gene family predates angiosperm evolution and suggests that Selaginella and Equisetum arose prior to Psilotum. J. Mol. Evol. 41:329-337. 4. Miller, J. S. 1988. Ultrafiltration for the removal of excess DNA linkers subsequent to ligation. BioTechniques. 5:632-634. 5. Sambrook, J., E. F. Frisch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual., 2nd ed. Cold Spring Harbor Publications, Cold Spring Harbor, NY. 6. Seeley, K. 1999. Purification and handling of DNA fragments, p. 4-10, Nanosep® Centrifugal Devices: Protocols for Use. Pall Life Sciences, Ann Arbor, MI. 7. Wang, H., M. Qi, and A. J. Cutler. 1993. A simple method of preparing plant samples for PCR. Nucleic Acids Res. 21:4153-4154. 8. Zeng, Q., M. K. Eidsness, and A. O. Summers. 1997. Near-zero background cloning of PCR products. BioTechniques. 23:412-414 Ordering Information Complementary Products Nanosep® Centrifugal Devices with Omega™ Membrane • AcroPrep™ 96- and 384-well Filter Plates can be used for a variety of molecular biology, combinatorial chemistry, Product No. Description Packaging and screening applications. OD003C33 3K, gray 24/pkg • AcroWell™ 96-well Filter Plates with BioTrace™ and OD003C34 3K, gray 100/pkg GHP Membranes can be used for hybridization-based assays such as dot blots, ELISAs and TRF. OD003C35 3K, gray 500/pkg • Vivid™ Gene Array Slides feature a unique membrane OD010C33 10K, blue 24/pkg construction that allows high signal-to-noise ratios, requires less template, and provides consistent results. OD010C34 10K, blue 100/pkg • BioTrace, Biodyne®, and FluoroTrans® Transfer OD010C35 10K, blue 500/pkg Membranes offer precise performance and compatibility OD030C33 30K, red 24/pkg with nearly every detection system available. OD030C34 30K, red 100/pkg • Stirred Cell Systems feature high performance ultrafiltration membranes ultrasonically sealed into 10 mL OD030C35 30K, red 500/pkg and 150 mL cells. OD100C33 100K, clear 24/pkg • Ultrafiltration Membrane Discs are highly porous, OD100C34 100K, clear 100/pkg providing fast flow rates and high recoveries. OD100C35 100K, clear 500/pkg OD300C33 300K, orange 24/pkg OD300C34 300K, orange 100/pkg OD300C35 300K, orange 500/pkg

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