Sp blood cell culture dna isolation kit

Manufactured by Bionano Genomics

Sourced in United States

The SP Blood & Cell Culture DNA Isolation Kit is a laboratory equipment product designed for the extraction and purification of genomic DNA from blood samples and cell culture. The kit utilizes a spin column-based method to efficiently isolate high-quality DNA for various downstream applications, such as PCR, sequencing, and analysis.

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Lab products found in correlation

Saphyr instrument, by Bionano Genomics (5 mentions) Dls direct label and stain dna labeling kit, by Bionano Genomics (4 mentions) Bionano solve version 3, by Bionano Genomics (1 mentions) Saphyr g2.3 chip, by Bionano Genomics (1 mentions) Prep direct label and stain dls protocol, by Bionano Genomics (1 mentions) Phenylmethylsulfonyl fluoride pmsf, by Merck Group (1 mentions) Solve v 3, by Bionano Genomics (1 mentions) Bionano access, by Bionano Genomics (1 mentions) Dls dna labeling kit, by Bionano Genomics (1 mentions)

6 protocols using sp blood cell culture dna isolation kit

Ultra-high Molecular Weight DNA Isolation

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For each sample, a minimum of 650 µL of whole peripheral EDTA blood was used to purify ultra-high molecular weight DNA using the SP Blood & Cell Culture DNA Isolation Kit (Bionano Genomics). Briefly, after counting, white blood cells were pelleted (2200 g for 2 min) and treated with LBB lysis buffer and proteinase K to release genomic DNA (gDNA). After inactivation of proteinase K by PMSF treatment, genomic DNA was bound to a paramagnetic disk, washed and eluted. Ultra-high molecular weight DNA was left to homogenise at room temperature overnight. The next day, DNA molecules were labelled using the DLS (Direct Label and Stain) DNA Labeling Kit (Bionano Genomics). Seven hundred fifty nanograms of gDNA were labelled in the presence of direct label enzyme (DLE-1) and DL-green fluorophores. After clean-up of excess DL-green fluorophores and rapid digestion of the remaining DLE-1 enzyme by proteinase K, the DNA backbone was counterstained overnight before quantification and visualisation on a Saphyr instrument. A volume of 8.5 µL of labelled gDNA solution of concentration between 4 and 12 ng/µL was loaded on a Saphyr chip and scanned on the Saphyr instrument (Bionano Genomics).

Scharf F., Leal Silva R.M., Morak M., Hastie A., Pickl J.M., Sendelbach K., Gebhard C., Locher M., Laner A., Steinke-Lange V., Koehler U., Holinski-Feder E, & Wolf D.A. (2021). Constitutional chromothripsis of the APC locus as a cause of genetic predisposition to colon cancer. Journal of Medical Genetics, 59(10), 976-983.

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Bionano Optical Genome Mapping for Rare Variants

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Bionano optical genome mapping was performed as described previously.³⁵ (link)^,³⁶ (link) In brief, DNA was isolated from a lymphoblastoid cell line obtained from an affected male from family A (III-8; Figure 1), according to the manufacturer’s instructions using the SP Blood & Cell Culture DNA Isolation Kit, (Bionano Genomics, San Diego, CA, USA). The isolated UHMW DNA was labeled for the CTTAAG sequence, using the DLS (Direct Label and Stain) DNA Labeling Kit (Bionano Genomics, San Diego, CA, USA), and was analyzed using a 3 × 1,300 Gb Saphyr chip (G2.3) on a Bionano Saphyr instrument, reaching 177× effective coverage with a label density of 14.37/100 kb and an average N50 of 232 kb. De novo assembly (using GRCh37) and variant annotation was performed using Bionano Solve version 3.4, which includes two different algorithms for SV (based on assembled maps) and copy number variant (CNV) (based on molecule coverage) calling. Annotated variants were filtered for rare events as described previously.³⁶ (link) In addition, the region of interest around exon 12 of CHM was analyzed visually in Bionano Access version 1.4.3.

Fadaie Z., Neveling K., Mantere T., Derks R., Haer-Wigman L., den Ouden A., Kwint M., O’Gorman L., Valkenburg D., Hoyng C.B., Gilissen C., Vissers L.E., Nelen M., Cremers F.P., Hoischen A, & Roosing S. (2021). Long-read technologies identify a hidden inverted duplication in a family with choroideremia. Human Genetics and Genomics Advances, 2(4), 100046.

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Optical Genome Mapping for Structural Variant Detection

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Optical genome mapping (Bionano Genomics, San Diego, CA, USA) was performed as previously described (Mantere et al. 2021 (link); Neveling et al. 2021 (link)). Ultra-high molecular weight DNA was isolated from whole peripheral blood (collected in EDTA tubes) using the SP Blood & Cell Culture DNA Isolation Kit (Bionano Genomics, San Diego, CA, USA). CTTAAG labeling was performed using the DLS (Direct Label and Stain) DNA Labeling Kit (Bionano Genomics, San Diego, CA, USA) and the labeled sample was analyzed using a 3 × 1300 Gb Saphyr chip (G2.3) on a Saphyr instrument (Bionano Genomics, San Diego, CA, USA). An effective coverage of 124 × was reached, with a label density of 14.63/100 kb and an average N50 of 279 kb. De novo assembly (using GRCh37 and GRCh38) and variant annotation were performed using Bionano Solve version 3.4, which includes two separate algorithms for SV and CNV detection. Annotated variants were filtered for rare events as described previously (Mantere et al. 2021 (link)). In addition, the genomic region spanning the CEVA haplotype was analyzed visually in Bionano Access version 1.4.3.

Smits J.J., de Bruijn S.E., Lanting C.P., Oostrik J., O’Gorman L., Mantere T., Cremers F.P., Roosing S., Yntema H.G., de Vrieze E., Derks R., Hoischen A., Pegge S.A., Neveling K., Pennings R.J, & Kremer H. (2021). Exploring the missing heritability in subjects with hearing loss, enlarged vestibular aqueducts, and a single or no pathogenic SLC26A4 variant. Human Genetics, 141(3-4), 465-484.

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Optical Genome Mapping for Rare Variant Detection

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OGM was performed as previously described [33 (link), 34 (link)]. In brief, high‐molecular‐weight germline DNA was extracted from an Epstein–Barr virus (EBV)‐transformed lymphoblastoid cell line from II‐2, using the SP Blood & Cell Culture DNA Isolation Kit according to the manufacturer's instructions (Bionano Genomics®, San Diego, CA, USA). The cell pellet (1.5 million cells) was resuspended in proteinase K and RNAse and mixed with LBB‐lysis buffer to release genomic DNA (gDNA). The sample was treated with phenyl‐methyl‐sulfonyl‐fluoride (PMSF) (Sigma‐Aldrich, St Louis, MO, USA), and isopropanol was added after placing a Nanobind disk on each sample. After washing, gDNA was eluted from the disks and equilibrated overnight to facilitate DNA homogeneity. Next, gDNA was labeled according to the manufacturer's instructions using the Bionano Prep Direct Label and Stain (DLS) Protocol. The sample was subsequently stained overnight using the Bionano DNA stain reagent and loaded on a 3 × 1300 Gb Saphyr chip (G2.3) to be imaged using the Saphyr instrument. Bionano Solve v3.4 was used to perform the de novo genome assembly, variant calling, and annotation with default settings. Annotated variants were filtered for rare events as described previously [34 (link)].

Sabatella M., Mantere T., Waanders E., Neveling K., Mensenkamp A.R., van Dijk F., Hehir‐Kwa J.Y., Derks R., Kwint M., O'Gorman L., Tropa Martins M., Gidding C.E., Lequin M.H., Küsters B., Wesseling P., Nelen M., Biegel J.A., Hoischen A., Jongmans M.C, & Kuiper R.P. (2021). Optical genome mapping identifies a germline retrotransposon insertion in SMARCB1 in two siblings with atypical teratoid rhabdoid tumors. The Journal of Pathology, 255(2), 202-211.

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Whole Genome Sequencing and Structural Variation Analysis

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Genomic DNA was isolated from peripheral blood lymphocytes following standard procedures and analyzed through genome sequencing as described previously. 6 Sequencing was performed by BGI on a BGISeq500 using a 2x 100 basepair (bp) or 2x 150 bp paired-end module, with a minimal median coverage per genome of 30 fold. Read mapping to the Human Reference Genome build GRCh38/hg38 and single-nucleotide variant (SNV) calling were performed using Burrows-Wheeler Aligner V.0.78 14 In brief, ultrahigh molecular weight DNA was isolated from whole peripheral blood (EDTA) using the SP Blood & Cell Culture DNA Isolation Kit (Bionano Genomics). DNA labeling was performed using the Direct Label and Stain (DLS) DNA Labeling Kit (Bionano Genomics), and the labeled sample was loaded on a 3×1300 Gb Saphyr chip (G2.3) on a Saphyr instrument (Bionano Genomics). Annotated de novo assembly using the genome build hg19 was performed using Bionano Solve version 3.6.1, which includes 2 separate algorithms for SV and CNV detection as described previously. 12 12. Mantere, T. • Neveling, K. • Pebrel-Richard, C. ...

de Bruijn S.E., Rodenburg K., Corominas J., Ben-Yosef T., Reurink J., Kremer H., Whelan L., Plomp A.S., Berger W., Farrar G.J., Ferenc Kovács Á., Fajardy I., Hitti-Malin R.J., Weisschuh N., Weener M.E., Sharon D., Pennings R.J.E., Haer-Wigman L., Hoyng C.B., Nelen M.R., Vissers L.E.L.M., van den Born L.I., Gilissen C., Cremers F.P.M., Hoischen A., Neveling K, & Roosing S. (2023). Optical genome mapping and revisiting short-read genome sequencing data reveal previously overlooked structural variants disrupting retinal disease-associated genes. Genetics in medicine : official journal of the American College of Medical Genetics, 25(3).

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Comparative Genomics of Great Apes

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High-molecular weight DNA from one chimpanzee (Pan troglodytes 15), one bonobo (Bonobo Banya), one gorilla (Gorilla gorilla 1), one orangutan (Pongo pygmaeus 8), and the rhesus macaque was extracted using the SP Blood & Cell Culture DNA Isolation kit (Bionano Genomics) and labeled using the DLS DNA labeling kit (DLE-1 labeling enzyme, Bionano Genomics). Samples were loaded onto Saphyr Chips G2.3 (Bionano Genomics), linearized, and visualized using the Saphyr Instrument (Bionano Genomics), according to the Saphyr System User Guide.
All analyses were performed in Bionano Access (Bionano Genomics). After general quality assessment via the Molecule Quality Report, a de novo assembly was performed against the most recent human reference genome hg38. Structural variants could be detected at the genome-wide level in the generated circos plot. The 22q11.2 region was visually inspected for structural rearrangements by zooming in to this region and comparing the compiled haplotypes with the hg38 reference.

Vervoort L., Dierckxsens N., Pereboom Z., Capozzi O., Rocchi M., Shaikh T.H., & Fryns J. (2020). Human-specific expansion of 22q11.2 low copy repeats.

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