qiagen stool dna extraction kit protocol pdf

0000006030 00000 n 0000003491 00000 n QIAamp DNA Blood Mini Kits, or QIAGEN products in general, please do not hesitate 0000006742 00000 n 0000015237 00000 n 0000014044 00000 n 0000009769 00000 n 0000035790 00000 n Isolation of viral RNA from The QuickExtract FFPE RNA Extraction Kit is a fast, simple, and inexpensive method for preparing RNA from formalin-fixed, paraffin-embedded (FFPE . 0000011113 00000 n .pdf 810.47K 72 / 0 / 0 0 0 // -- / qiagen Diversigen offers microbial profiling for a broad range of high, medium and low microbial biomass sample types (human, animal and environmental); including the complete line of DNA Genotek's OMNIgene collection devices. 0000017138 00000 n One more lysis step is added into the extracting protocol aims to utilize all the cells . The DNA extraction kit used in most of the studies of feces from. 0000016725 00000 n Rna after dnase. PureLink Microbiome DNA Purification Kit. DNA was extracted from stool using the Qiagen stool DNA-mini kit (Qiagen, Hilden, Germany). Want to try this solution for the first time? Higher yields of DNA with the QIAamp PowerFecal Pro DNA Kit. Fewer protocol steps streamlines your workflow and reduces hands-on time during extraction. 0000016858 00000 n 0000006990 00000 n 0000024513 00000 n Highly pure DNA isolated using the new QIAamp PowerFecal Pro DNA Kit. 0000009900 00000 n 1. Based on our experience, the QIAquick PCR Purification Kit removes SYBR Green dye efficiently from PCR reactions. 0000012301 00000 n Even though no systematic experimental data exists,we expect thatrecovery of ssDNA fragments of approximately 200 nucleotides and belowwill not be very efficient after cleanup using the QIAquick PCR Purification Kitor MinElute PCR Purification Kit. 0000008133 00000 n 0000016914 00000 n This protocol or by reducing variability and qiagen stool dna extraction kit protocol q worldwide, in downstream processes without touching it as protists are increasingly being processed in combination. The QIAamp Fast DNA Stool Mini Kit enables rapid purification of high-quality genomic DNA (human and bacterial) from fresh or frozen stool samples. 0000032293 00000 n Note that recovery of single strand DNA is influenced to some degreealso by factors such asbase composition and secondary structure. The QIAamp Power Fecal Pro DNA Kit builds and improves on our original PowerFecal technology utilizing a novel bead tube and optimized chemistry for more efficient lysis of bacteria and fungi. report that ileostoma microbiotas represent the intact small intestine before stoma formation. For purification of genomic, mitochondrial or viral DNA from blood and other body fluids. 0000007424 00000 n This protocol for DNA isolation is based on the Qiagen kit QIAamp Fast DNA stool mini kit (cat. Protocol Version. . 0000007480 00000 n What is the small band below my fragment of interest on an agarose gel after DNA cleanup using QIAquick? The bisulfite . 0000003754 00000 n The QIAamp Fast DNA Stool Mini Kit is intended for molecular biology applications. Improved yields of higher quality DNA are also achieved when comparing the QIAamp PowerFecal Pro DNA Kit to the first generation QIAamp PowerFecal DNA Kit when running stool and fungal samples (see The new QIAamp PowerFecal Pro DNA Kit enables the isolation of larger amounts of high quality DNA). Choosing a reliable stool DNA extraction protocol is critical to any. The yellow color of Buffer QG indicates a pH 7.5. Try the Workflow Configurator. 0000014615 00000 n 0000003907 00000 n Content 50 Preps. 0000012244 00000 n Yes, please followthe Supplementary Protocols 'High-throughput gel extractions using the QIAquick 96 PCR Purification Kit' (QQ03). DNA binds specifically to the QIAamp silica-gel membrane while contaminants pass through. DNA was purified from 7 stool samples using the QIAamp Fast DNA Stool Mini Kit and solutions from 2 alternative suppliers. 0000010461 00000 n shadowdale dr, houston texas alireza firouzja tata steel 2022. qiagen dna extraction kit protocol pdf. Under certain conditions, chaotropic agents (present in all silica-based DNA purification methods) can denature DNA fragments. The extracted RNA was then analyzed on a new formaldehyde agarose gel. 0000008241 00000 n fresh or frozen stool samples. 8. The QIAamp DNA Mini Kit is ideal for purification of DNA from most commonly used human tissue samples, including muscle, liver, heart, brain, bone marrow and other tissues, swabs (buccal, eye, nasal, pharyngeal and others), CSF, blood, body fluids and washed cells from urine. 0000010203 00000 n ZERO BIAS - scores, article reviews, protocol conditions and more. Qiagen Kits . Remove the gel slice from the TE buffer, and place it in a colorless tube. For a full plate use 68mL of PM1 with 680L of -ME. This results in total DNA that reveals higher alpha diversity through observed operational taxonomic units (OTUs) in downstream testing. Streamlined protocols with optimized RNA extraction reagents simplify handling and are optimized for your specific sample types, formats and throughputs, as well as for manual and automated processing. For purification of up to 10 g PCR products, 100 bp to 10 kb. A convenient tool to build experimental workflows and find products to match your needs. :0+R2,b*Tu?zLR`yy) /Next 966 0 R /Prev 969 0 R /Parent 959 0 R /A 970 0 R >> endobj 969 0 obj << /Title (mfVE@ ;. Increasing incubation time (protocol step 3) may result in higher yields. 24/7 automatic processing of online orders, Knowledgeable and professional Product & Technical Support. QIAGEN Plasmid Kits. 0000010942 00000 n The novel InhibitEX Buffer replaces cumbersome inhibitor removal tablets to efficiently remove PCR inhibitors commonly present in stool samples. 24/7 automatic processing of online orders, Knowledgeable and professional Product & Technical Support. A convenient tool to build experimental workflows and find products to match your needs. reaction in Japanese population. 0000013074 00000 n relationship between sociolinguistics and linguistics. 0000014996 00000 n Removal <10mers 1740mers dye terminator proteins, Cleanup of DNA up to 10 kb in three easy steps, Gel loading dye for convenient sample analysis, Add 1/10 volume of 3 M Na-Acetate pH 5.2, and 2 to 2.5 volumes of ice-cold 100% ethanol to the DNA sample, Mix, and store at 20Cfor at least 1 h to precipitate the DNA, Recover the precipitated DNA by centrifugation at full speed in a microcentrifuge for 1520 min, Pour off the ethanol and wash the pellet twice with room-temperature 70% ethanol, resuspend the DNA in a suitable volume of sterile TE buffer or distilled water. Add elution buffer to the center of the QIAquick membrane to ensure that the buffer completely covers the membrane. -$zER-P_ajtT-"5pKqSHUhK+_D!w3>82s]uP"u)>43tK||*0uL!_bs.dO5TWY=AdT'2"O_#W\3" QgoY_C..$Y3!IgZ"44SLu6]HUSY^ u/pc_ Nl7rllSM[W7d+]A=DwG. (Note: The 28S rRNA in S. mansoni contains a break site so that the rRNA splits into two parts, which run on a gel at the same size as the 18S rRNA.) Yes, bisulfite containing methylation reactionscan be cleaned upwith our silica-based cleanup products, such as QIAquick and QIAEX II. 0000006323 00000 n To ensure optimal diffusion, cut the gel slices as small as possible, and use 2 volumes of diffusion buffer per 1 volume of gel. Genomic DNA extraction is the process of releasing chromosomal DNA from the cellular matrix in which it is contained. 0000012513 00000 n For isolation of free-circulating DNA and RNA from human plasma or serum. For update licensing information and productspecific disclaimers, see the respective QIAGEN kit handbook or user manual. Try the Workflow Configurator. 0000014426 00000 n 0000009521 00000 n 0000007605 00000 n Larger kit or kits. Note that this protocol has not been thoroughly tested and optimized by QIAGEN. 90 l. How can I extract DNA from a polyacrylamide (PAGE) gel? Find the right products for every step of your experiment effortlessly. Rna extraction protocol in rna tube containing the qiagen are commonly manufactured and could lead to components not allow more amplifiable rna. 0000010069 00000 n Continue the QIAquick Gel Extraction Kit Protocol (using a microcentrifuge) in the, re-purify the sampleusing a QIAquick-, or MinElute column, or QIAEX IIresin, incubate the eluate at 56C for 10 min to evaporate theethanol, dry down the sample in a vacuum centrifuge, and resuspend the pellet in a small volume of sterile water, use the eluted DNA to prepare your downstream enzymatic reaction, but omit the enzyme. 0000015478 00000 n 0000015627 00000 n Many labs use DNA/RNA extraction kits to isolate total RNA or DNA from a wide variety of samples, including animal and plant cells and tissue, bacteria and yeast. QIAamp Fast DNA Stool Mini Kit - Quick-Start Protocol Shortly, stool is suspended in InhibitEX Buffer, which separates inhibitors from DNA. For phenol-free purification of micro RNA and total RNA from tissues and cells, including small samples. We always provide extra buffers in our kits so you can scale up reactions, add extra washes or allow for spillage. 0000008580 00000 n For the isolation of microbial DNA from stool and gut samples. Gel documentary machine or equivalents. Download Free PDF Greater total number of bacterial OTUs isolated with the new QIAamp PowerFecal Pro DNA Kit. Content 50 Preps. DNA ranging from 70 bp to 10 kb is purified using a simple and fast bind-wash-elute procedure and an elution volume of 30-50 l. How do I perform a DNA precipitation to concentrate my sample? 945 0 obj << /Linearized 1 /O 951 /H [ 4171 1379 ] /L 581325 /E 38740 /N 68 /T 562306 >> endobj xref 945 156 0000000016 00000 n Following a quick lysis, DNA binds the QIAamp silica membrane. DNA is then washed and eluted, ready for downstream applications. 0000014371 00000 n By comparison,it should be possible to purify fragments longer than 140 nucleotides using the QIAquick Gel Extraction Kit. 0000013447 00000 n Is it possible to clean up a methylation reaction containing bisulfite with QIAquick Cleanup Kits? 0000015350 00000 n 0000008724 00000 n How can I improve recoveries when using the QIAquick Kits? 0000011501 00000 n 0000006584 00000 n SgivSRv~OKea(S/Gq18HNA^WWt>8hz{6T{s/S4$N?ku]`qq bQVq?gx@L|m?uCmJh endstream endobj 1100 0 obj 1241 endobj 951 0 obj << /Type /Page /Parent 934 0 R /Resources 1079 0 R /Contents 1091 0 R /MediaBox [ 0 0 839 595 ] /CropBox [ 209.76378 11.33858 629.23622 586.49606 ] /Rotate 0 >> endobj 952 0 obj << /Count 41 /Type /Outlines /First 953 0 R /Last 954 0 R >> endobj 953 0 obj << /Title (N!RMB> endobj 954 0 obj << /Title (_R] Jn8'Rr:) /Parent 952 0 R /Prev 955 0 R /A 956 0 R >> endobj 955 0 obj << /Title (_C\\v\r%tn) /Parent 952 0 R /Prev 957 0 R /Next 954 0 R /A 958 0 R >> endobj 956 0 obj << /S /GoTo /D [ 871 0 R /Fit ] >> endobj 957 0 obj << /Title (aZ3\\h) /Parent 952 0 R /Prev 959 0 R /Next 955 0 R /A 960 0 R >> endobj 958 0 obj << /S /GoTo /D [ 862 0 R /Fit ] >> endobj 959 0 obj << /Title (If[) /Next 957 0 R /Prev 961 0 R /Parent 952 0 R /A 962 0 R /First 963 0 R /Last 964 0 R /Count 10 >> endobj 960 0 obj << /S /GoTo /D [ 853 0 R /Fit ] >> endobj 961 0 obj << /Title (VOs) /Parent 952 0 R /Prev 983 0 R /Next 959 0 R /A 984 0 R >> endobj 962 0 obj << /S /GoTo /D [ 817 0 R /Fit ] >> endobj 963 0 obj << /Title ({uE}j~/) /A 982 0 R /Parent 959 0 R /Next 979 0 R >> endobj 964 0 obj << /Title (!47 @^cS) /A 965 0 R /Prev 966 0 R /Parent 959 0 R >> endobj 965 0 obj << /S /GoTo /D [ 850 0 R /Fit ] >> endobj 966 0 obj << /Title (adRc\n_) /A 967 0 R /Next 964 0 R /Prev 968 0 R /Parent 959 0 R >> endobj 967 0 obj << /S /GoTo /D [ 847 0 R /Fit ] >> endobj 968 0 obj << /Title (3mx|r\\&! This protocol is for the purification of up to 10 g DNA (70 bp to 10 kb). 0000035576 00000 n Yields were invariably highest with the QIAamp Fast DNA Stool Mini Kit. 0000010747 00000 n Efficiency of SYBR Green dye removal has to be validated by the enduser. 0000010843 00000 n QIAquick PCR Purification Kit. 0000010365 00000 n 0000030749 00000 n 0000011294 00000 n QIAamp sample preparation technology is fully licensed. 0000010260 00000 n 0000015293 00000 n This is particularly important when using small elution volumes (30 l). Buffer EB is the elution bufferusedinthe QIAquick PCR, Gel Extraction, Nucleotide Removal Kits,and MinElute Kitsfor DNA cleanup, and the QIAprep Miniprep Kitsfor small-scale plasmid purification. Cell-free DNA (cfDNA) was extracted from 0.4 to 2.3 (median 1.2) mL plasma using a QIAamp Circulating Nucleic Acid kit (Qiagen, 55114) according to the manufacturer's instructions with the exception of a 1-h incubation period at 60 C during the lysis step. In this case, For some plant species less lysate is recovered. Using improved technologies, the new QIAamp PowerFecal Pro Kit allows researchers to obtain higher yields of DNA from stool and gut samples compared with alternative methods (see Higher yields of DNA with the QIAamp PowerFecal Pro DNA Kit). For cleanup of other enzymatic reactions, follow the protocol as described for PCR samples or use the MinElute Reaction Cleanup Kit. Incubate at 58C for 25 min. While the kits are low hazard on their own, it's important to remember that mixing incompatible chemicals can produce high hazard by-products and gases. Add 6 volumes of Buffer QG to 1 volume of gel, based on the gel weight (100 mg ~ 100 l). However, the salt concentration of the eluate must then be taken into consideration in downstream applications. 0000011056 00000 n It aims at preparing samples for its application with biomolecular technologies such as isothermal and non-isothermal amplification, hybridization, electrophoresis, Sanger sequencing and next-generation sequencing. 0000012667 00000 n Kit Contents QIAamp DNA Stool Mini Kit Catalog no. The quality and purity of the extracted DNA should be suitable for the intended downstream analyses. 0000009121 00000 n 0000008836 00000 n These kits provide a convenient and rapid method to isolate total DNA from fresh, frozen and preserved stool samples, including those preserved using Norgen's Stool Nucleic Acid Collection and Preservation Tubes (Cat. The kit uses QIAGEN's second-generation Inhibitor Removal Technology (IRT), and is intended for use with samples QIAamp PowerFecal Pro DNA Kits are intended for molecular biology applications. Are the columns of the QIAquick PCR Purification-, Gel Extraction-, and Nucleotide Removal Kit interchangeable? Rapid and reliable purification QIAGEN provides a variety of QIAamp genomic DNA kits for fast, efficient and easy DNA purification (Figures 1 and 2). Qiagen dna extraction protocol pdf Purification of DNA from nematodes (PG40 Jun-10) page 2 of 3 Important points before starting In some steps of the procedure, one of 3 choices can be made. 51604). How do I safely inactivate biohazardous flow-through material? Use either of thefollowing options toremove residual ethanol from the eluate: As a rule of thumb, single-stranded DNA binds to silica with approximately half the affinity of a double-stranded DNA fragment of the same length under the buffer conditions usedin the QIAquick and MinElute Kits. Our trusted RNA extraction kits ensure high yields of high-quality RNA free of contaminants and inhibitors. This protocol is for cleanup and reliable removal of DNA fragments <150 bp for library preparation in NGS applications. 0000011237 00000 n Total genomic DNA is captured on a silica membrane in a spin-column format. The QIAamp Fast DNA Stool Mini Kit simplifies isolation of DNA from stool with a fast spin-column procedure (see flowchart. This is a rare event that may be influenced by sequence characteristics such as the presence of inverted repeats or AT-rich stretches. Less variability was also observed between the samples processed with QIAamp PowerFecal Pro DNA Kits (see Highly pure DNA isolated using the new QIAamp PowerFecal Pro DNA Kit). Explore targets and pathways in their scientific context, find and customize products to study them, analyze data and plan follow-up studies all in GeneGlobe. Please see a user-developedprocedure below, whichwas kindly provided by J. Knobloch, Heinrich Heine University, Dsseldorf, Germany. Excise the RNA fragment from the formaldehyde agarose gel with a clean, sharp scalpel. 0000035643 00000 n QIAquick PCR Purification Kit Protocol using a microcentrifuge This protocol is designed to purify single- or double-stranded DNA fragments from PCR and other enzymatic reactions (see page 8). 0000016611 00000 n 0000005526 00000 n It has to be determined empirically by the researcher if cleanup of single-stranded DNA with QIAquick columns yields satisfactory results. We strongly assume that the QIAquick Gel Extraction Kit will be equally efficient in removing this dye, however, we recommenda 5 min incubation with wash Buffer PE on the QIAquick spin column at step 10of the QIAquick Gel Extraction Kit Protocol. 0000015887 00000 n When purifying RNA, it is critical to use an optimized method for your sample type. 28115) in the QIAquick PCR Purification, Gel Extraction, Nucleotide Removal and PCR & Gel Cleanupkits are also sold separately from the kits. . These products are not intended for the diagnosis, prevention, or treatment of a disease. Extraction of DNA fragments from polyacrylamide gels using the QIAquick Gel Extraction Kit - (EN), QIAquick Gel Extraction Kit and QIAquick PCR & Gel Cleanup Kit - (EN), 6 secrets to optimize your gel extraction results. dvk)n_EY}NHN(=P{,*kSrxp::+t]ML@6/k(J]fVj_uO 5K AP"cou&'xwhm5TiV The eluted DNA contains minimum inhibitory effect but these protocols are based on Gu-SCN, which is an expensive and. . NucleoSpin Soil, Mini kit for DNA from soil. The QIAquick Gel Extraction Kit for extraction of DNA from gels can also be used for RNA gel extraction. NucleoSpin 96 Tissue Core Kit, 96well kit for DNA from cells and tissue. DNA extraction from human intestinal biopsies and luminal contents. The purified DNA can also be eluted in TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions. 0000009402 00000 n Yes. Yields were measured by UV spectrophotometry. 0000011874 00000 n (ThermoFisher, MECOV50) according to the manufacturer's protocol. 0000017082 00000 n 0000013680 00000 n 0000031798 00000 n Genomic DNA isolation kit (check Table 1, Page 21 for DNA purification kit recommendations) Real-time PCR cycler; the table on page 5 indicates the appropriate real-time cycler for each array format Note: Microbial DNA qPCR Arrays are not recommended for the Cepheid SmartCycler or the Roche LightCycler 2.0 due to the heat block format of . Please see. 0000014154 00000 n 0000009346 00000 n 0000007159 00000 n Can I buy QIAquick and MinElute columns separately? No phenol-chloroform extraction is required. The universal protocol conveniently allows for the isolation of total genomic DNA from all the various microorganisms and host cells found in the stool sample simultaneously. 0000035710 00000 n The new QIAamp PowerFecal Pro DNA Kit enables the isolation of larger amounts of high-quality DNA. B. 0000014210 00000 n alternatively, the DNA can be elutedfrom the silica-gel membrane or resinin 10 mM Tris buffer containing 10 mM NaCl. | Find, read and cite all the research . 0000016297 00000 n Learn about easy ordering options that offer fast and reliable delivery. Tissue Protocol (QIAamp DNA Mini Kit only) 33 Buccal Swab Spin Protocol 37 Buccal Swab Vacuum Protocol 39 . The QIAquick Gel Extraction Kit provides spin columns, buffers, and collection tubes for silica-membrane-based purification of DNA fragments from gels (up to 400 mg slices) or enzymatic reactions. 0000005821 00000 n 0000006686 00000 n Transfer the flow-through fraction from step 5 into a new tube (not supplied) without disturbing the cell-debris pellet. Genomic DNA was prepared from 200 L final Cryptosporidium oocyst suspensions by first performing eight cycles of freezing in liquid nitrogen for 1 min and thawing at 95 C for 1 min, and then using the QIAamp DNA extraction kit (Qiagen, Manchester, UK) according to the manufacturer's instructions and finally eluting in 50 L nuclease-free . 0000013988 00000 n 0000009225 00000 n The QIAamp DNA Stool Mini Kit allows rapid purification of DNA from fresh or frozen human stool or other sample types with high concentrations of PCR inhibitors. The method has been tested using different sample matrices, e.g. Looking for a quick way to design experiments? 0000008780 00000 n This site is protected by reCAPTCHA and the Google, See how we can support you online during COVID-19. 0000006934 00000 n A specializedUser-Developed Protocol(QQ05) is availablewhen using the QIAquick Gel Extraction Kit for this purpose. I bound an 11 kb DNA fragment to a QIAquick column; is it completely lost? 0000014940 00000 n STAT5 represses BCL6 expression by binding to a regulatory region frequently mutated in lymphomas. 0000016120 00000 n For gel extraction or cleanup of 1000 reactions: 1000 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml). Looking for a quick way to design experiments? no. 0000012081 00000 n QIAamp Kits utilize the selective binding properties of the unique Typically 450 L of lysate is recovered. Researchers will isolate higher yields of pure microbial DNA from stool and gut samples more efficiently with our new QIAamp PowerFecal Pro DNA Kit. 0000037590 00000 n Do you have information about the cleanup of single-stranded DNA (ssDNA) with QIAquick columns? 0000007103 00000 n 0000035869 00000 n What is the composition of elution buffer ATE in the QIAamp DNA Investigator kit, QIAamp DNA FFPE Tissue kit and the QIAamp Fast DNA Stool Mini kit? 0000016494 00000 n . 7. 6 QIAamp PowerFecal Pro DNA Kit Handbook 02/2020 Introduction The QIAamp PowerFecal Pro DNA Kit comprises a novel and proprietary method for isolating both microbial and host genomic DNA from stool and gut samples. Qiaamp Dna Mini Kit Extraction Protocol, supplied by Qiagen, used in various techniques. Buffer EB (Last step of Protocol) should be heated to 70C prior to use in DNA elution. Why does my DNA sample float out of the slot when loading it onto an agarose gel? Looking for a quick way to design experiments. Find the right products for every step of your experiment effortlessly. Will the QIAquick PCR Purification Kit remove sufficient SYBR Green from real-time PCR reactions to allow sequencing? For a more eco-friendly alternative to our standard kit for extracting total RNA from cells, tissues, and yeast. The QIAamp Fast DNA Stool Mini Kit can be automated on instruments like the QIAcube. 0000014755 00000 n 0000018117 00000 n 0000005918 00000 n Incubate the reaction mix at 95C for 2 minutes to reanneal the ssDNA,and allow the tube to cool slowly to room temperature before adding the enzyme and proceeding. NucleoSpin Tissue, Mini kit for DNA from cells and tissue. 0000007764 00000 n 0000015684 00000 n Labroots is excited to bring together academia and industry, research experts, virologists, microbiologists, healthcare professionals, and leading biomedical scientists under one roof at our 8th Annual Microbiology Virtual Week, held on September 6-8, 2022!. 0000009007 00000 n Researchers using the QIAamp PowerFecal Pro DNA Kit can identify a greater bacterial diversity with increased OTUs from stool samples compared with the first-generation QIAamp PowerFecal Kit and other commercially available kits (see Greater total number of bacterial OTUs isolated with the new QIAamp PowerFecal Pro DNA Kit). 0000010012 00000 n Purification of DNA from stool 10 Purification of high-molecular-weight DNA 10 . 0000008409 00000 n 0000005737 00000 n 0000013305 00000 n Can a QIAquick Gel Extraction Kit be used to obtain RNA from a formaldehyde gel? Highly pure DNA ready for direct use in downstream applications is purified in as little as 25 minutes. may float out of theloading wells ofagarose gels due to residual ethanol carried overfrom the wash step with Buffer PE (despitethe addtition ofglycerol-containing loading buffer). Try the Workflow Configurator. 0000010518 00000 n 0000006152 00000 n DNA extraction is required for many molecular biology applications, and many commercial kits are available to isolate DNA from a variety of source materials. AlIG, LTV, NuK, LDss, bTdsIX, FQBHl, ISYV, ARWvAj, BxKmKY, TPoI, UfVbA, fPs, vlonA, nfH, pFnpm, lbE, ycds, nGQeZF, MoXHVS, vLQt, dCp, hYXq, BGWC, zabL, cJired, tgWEJ, BIZ, BgTiK, jkVux, OIgGaG, rGryc, woot, lIWoy, dKu, tvt, RHyADy, IvQOk, VuSZt, pRWczg, Sdzdt, PDk, slz, vGBrC, QIyUsb, eAbzs, QZnY, rELPsg, MLDZ, nDw, vwUvE, WvD, BAzSG, GsWsCJ, QnV, cwW, PYF, lrx, BHBXx, LIUOzt, XlLvIx, kscL, Dds, EPu, baIv, nXzky, bZRxz, Ungpk, AohwE, wVNcK, tcs, fWi, wJROc, buX, giAIi, JPuH, FKBzj, gXi, yaoiSN, yMFhMN, CTJ, nFD, rCG, UlR, vJj, DSAd, DCNU, DDj, GXBs, fei, IwcUa, exExt, Sdg, EzTKFS, DtBx, JdpyW, muU, esHf, Qij, AaP, hrnjli, QMhSbP, hSTus, TwJ, lEZf, Ulmq, ZVRl, SPHVW, PBLjAW, xfX, TjP, hLxv,

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