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Proteinase k digestion

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Proteinase K is a broad-spectrum serine protease used for the digestion and lysis of proteins. It is commonly used in molecular biology and biochemistry applications to degrade proteins during DNA and RNA extraction and purification procedures.

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Lab products found in correlation

Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions) Dna stain reagent, by Bionano Genomics (2 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Bionano prep direct label and stain dls protocol, by Bionano Genomics (1 mentions) Prep direct label and stain dls protocol, by Bionano Genomics (1 mentions) Yoyo 1, by Thermo Fisher Scientific (1 mentions) Taq ligase, by New England Biolabs (1 mentions) Irysprep reagent kit, by Bionano Genomics (1 mentions) Direct label enzyme method, by Bionano Genomics (1 mentions) Qubit dsdna hs kit, by Thermo Fisher Scientific (1 mentions) Taq polymerase, by New England Biolabs (1 mentions) Axioscope 5 microscope, by Zeiss (1 mentions) Mirneasy ffpe kit, by Qiagen (1 mentions) Prep direct label and stain, by Bionano Genomics (1 mentions)

Close spelling variants of this product

Proteinase K (758 citations) Proteinase K digestion buffer (2 citations)

7 protocols using proteinase k digestion

1

Chromosome Conformation Capture (3C) Assay

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Chromosome Conformation Capture (3C) assays were performed as we previously described60 (link). Briefly, cells were counted and balanced to the same number (6 million) before the 3C experiments to allow for comparison between different cell types or treatments. Cells were cross-linked and lysed. Chromatin was digested using 400 units of HindIII, followed by ligation with 4,000 units of T4 DNA ligase (NEB M0202S). Cross-links were reversed by Qiagen proteinase K digestion. 3C products were purified by phenol-chloroform extraction, followed by quantitative PCR (qPCR). To control for random digestion, ligation and different primer efficiencies, randomly ligated DNA fragments within the tested loci were generated as previously described61 (link)–68 (link). A standard curve for the Ct value of each 3C primer pair, anchor and bait, were generated from these randomly ligated DNA fragments. The 3C frequency of each primer pair was normalized to their corresponding standard curves and was further normalized to a loading control, primers hybridized to the genomic region of the RHO gene. Primers used are listed in Supplementary Table 2.
Bailey S.D., Zhang X., Desai K., Aid M., Corradin O., Cowper-Sal·lari R., Akhtar-Zaidi B., Scacheri P.C., Haibe-Kains B, & Lupien M. (2015). ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters. Nature Communications, 6, 6186.
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2

UHMW gDNA Fluorescent Labeling

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Fluorescent dye labeling of the UHMW gDNA (specific labeling and whole DNA staining) was performed according to the manufacturer’s guidelines (Bionano Prep Direct Label and Stain (DLS) Protocol, Bionano Genomics, #30206). A total of 750 ng UHMW gDNA was labeled specifically at sequence CTTAAG with DL-green fluorophores using the DLE-1 enzyme. Proteinase K digestion (Qiagen, Hilden, Germany) was performed thereafter, followed by removing DLE-1 remnants with two different membranes on a microplate. As the next step, the whole gDNA was labeled by using DTT and DNA blue fluorescent stain (Bionano Genomics DNA stain reagent). The samples were rotated on the HulaMixer for 1 h, followed by incubation overnight at RT and protection from light. The labeled UHMW gDNA was quantified using the QubitTM dsDNA HS Assay kit (ThermoFisher Scientific, Waltham, MA, USA). To ensure homogeneity, specimens were collected at two different locations (e.g., top and bottom or left and right side) from each sample tube. A concentration in the range of 4 to 12 ng/µL was classified as acceptable with a coefficient of variation of the specimen < 0.25. Thereafter, the stained and labeled UHMW gDNA was loaded on a Saphyr G2.3 chip. By the Saphyr device, the DNA molecules were imaged with a maximum capacity of 1500 Gbp per sample.
Suttorp J., Lühmann J.L., Behrens Y.L., Göhring G., Steinemann D., Reinhardt D., von Neuhoff N, & Schneider M. (2022). Optical Genome Mapping as a Diagnostic Tool in Pediatric Acute Myeloid Leukemia. Cancers, 14(9), 2058.
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3

Bionano Saphyr Chip DNA Labeling

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The labeling of the UHMW gDNA was done according to the manufacturer’s guidelines by using the Bionano Prep Direct Label and Stain (DLS) Protocol. Purified UHMW gDNA (750 ng) was first labeled with DL-green fluorophores using the DLE-1 enzyme. Next, Proteinase K digestion (Qiagen, Hilden, Germany) was performed and DLE was cleaned up using two different membranes on a microplate. The backbone of the DNA was labeled using DTT and DNA stain (DNA stain reagent, Bionano Genomics, San Diego, CA, USA). Then, the sample was rotated on the HulaMixer followed by overnight incubation at room temperature and protected from light. Two samples of each labeled gDNA taken from two different locations within the tube (left and right side) were quantified using the QubitTM dsDNA HS Assay kit (ThermoFisher Scientific, Waltham, MA, USA). The recommended DNA concentration is 4–12 ng/μL and a CV between the sampling <0.25. The labeled gDNA was loaded on the Saphyr G2.3 chips and molecules were imaged by the Saphyr instrument with a maximum capacity of 2000 Gb per sample.
Lühmann J.L., Stelter M., Wolter M., Kater J., Lentes J., Bergmann A.K., Schieck M., Göhring G., Möricke A., Cario G., Žaliová M., Schrappe M., Schlegelberger B., Stanulla M, & Steinemann D. (2021). The Clinical Utility of Optical Genome Mapping for the Assessment of Genomic Aberrations in Acute Lymphoblastic Leukemia. Cancers, 13(17), 4388.
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4

Optical Genome Mapping with Bionano

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The DNA was labeled with nick-labeling [47 (link)] as described previously using the IrysPrep Reagent Kit (BioNano Genomics). Specifically, 300 ng of purified genomic DNA was nicked with 7 U nicking endonuclease Nt.BspQI (New England BioLabs, NEB) at 37°C for two hours in NEB Buffer 3.1. The nicked DNA was labeled with a fluorescent-dUTP nucleotide analog using Taq polymerase (NEB) for one hour at 72°C. After labeling, the nicks were ligated with Taq ligase (NEB) in the presence of dNTPs. The backbone of fluorescently labeled DNA was stained with YOYO-1 (Invitrogen).
A subset of the samples were labeled using the newer Direct Label Enzyme (DLE) method (Bionano Genomics). These samples used the DNA Labeling Kit-DLS 80005 and followed the manufacturer’s instructions. In Summary, 750ng of the gDNA was labeled using DLE-1 enzyme and reaction mix followed by Proteinase K digestion (Qiagen). The DNA back bone was stained after drop dialysis. The stained sample was then homogenized and incubated at room temperature over nigh before quantified using Qubit dsDNA HS Kit (Invitrogen).
Young E., Abid H.Z., Kwok P.Y., Riethman H, & Xiao M. (2020). Comprehensive Analysis of Human Subtelomeres by Whole Genome Mapping. PLoS Genetics, 16(1), e1008347.
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5

RNA Isolation and Expression Analysis from FFPE Breast Cancer Tissues

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Prior to RNA isolation, the BC tissues were microdissected from 505 samples. Microdissection was performed by a pathologist using a Zeiss axioscope 5 microscope (Zeiss, Germany) and tumor tissue was marked with a pencil to exclude pre-existing breast parenchyma. For RNA isolation, two to four 10-μm-thick tissue slides were used. Deparaffinization of FFPE tissues was performed with 2 × xylene for 5 min, followed by incubation in 96% and 70% ethanol for 2 min each. Proteinase K digestion (Qiagen, cat#19131) was performed for up to 2 h at room temperature followed by 15 min at 80°C. RNA was isolated with a Qiagen miRNeasy FFPE Kit (Qiagen, cat#217504) according to the manufacturer’s instructions. RNA expression analysis was performed using the NanoString nCounter XT Assay according to the Hybridization protocol for the nCounter XT CodeSet Gene Expression Assay (NanoString nCounter, Seattle, WA, USA) and data analysed with the nCounter Expression Data Analysis Guide (MAN- C0011–04 from 2017). From each sample, 400 ng of FFPE-derived RNA were used. All steps were performed as described in nCounter XT Assay User Manual (MAN-10023–11 from 2016). For the analysis a custom codeset design was used and all respective genes are summerized in Supplementary Table S3. The expression levels were evaluated with the NanoStringNorm (https://github.com/sgrote/NanoStringNormalizeR/) R package.
Ullu E, & Tschudi C. (1991). Trans splicing in trypanosomes requires methylation of the 5' end of the spliced leader RNA.. Proceedings of the National Academy of Sciences of the United States of America, 88(22), 10074-10078.
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6

Quantifying Viral RNA in Dengue and West Nile Virus

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Viral RNA was extracted from DENV-2 and WNV that were serially diluted (10 to 5 × 103 PFU/ml) in PBS followed by a one-step qPCR assay. Viral RNA was isolated by proteinase K digestion (Qiagen) and AL lysis (Qiagen) of viral particles, followed by RNA purification using the RNase easy isolation kit (Qiagen). The A one-step qPCR method was performed using previously reported primers and probes for the detection of DENV-2 capsid and WNV envelope(E) gene31 (link)–32 (link), and were purchased either by Integrated DNA Technologies (Coralville, IA) or Applied Biosystems (Grand Island, NY). All results were expressed as the absolute number of viral RNA copies/100 µl of sample using the iTAQ™ Universal Probes one-step qPCR kit (Bio-Rad, Hercules, CA) and were compared to viral gene standards for absolute copy number quantification.
Paul A.M., Fan Z., Sinha S.S., Shi Y., Le L., Bai F, & Ray P.C. (2015). Bio-Conjugated Gold Nanoparticle Based SERS Probe for Ultrasensitive Identification of Mosquito-Borne Viruses Using Raman Fingerprinting. The journal of physical chemistry. C, Nanomaterials and interfaces, 119(41), 23669-23775.
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7

UHMW DNA Labeling and Quantification

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The UHMW gDNA labelling was performed following the manufacturer's guidelines using the Bionano Prep Direct Label and Stain (DLS) Protocol. Briefly, 750ng purified UHMW DNA was labelled with DL-green fluorophores using the Direct Labeling Enzyme (DLE-1) chemistry, followed by Proteinase K digestion (Qiagen) and DL-green cleanup using two membrane adsorption steps on a microplate. Finally, the labelled samples were homogenized by mixing with HulaMixer and stained overnight (Bionano DNA stain reagent) at room temperature, protected from light, to visualize the DNA backbone.
DNA quantification was carried out using Qubit dsDNA assay HS kit with a Qubit 3.0 Fluorometer (ThermoFisher Scientific). 40/48 (83.3%) of the labelled samples had concentrations within the recommended values of 4-12 ng/μl for both measurement points, 7/48 samples had one measurement >12 ng/μl, and only one sample had both measurements below 4 ng/μl (Supplementary Table 1).
Neveling K., Mantere T., Vermeulen S., Oorsprong M., Beek R.v., Kater‐Baats E., Pauper M., Zande G.v.d., Smeets D., Weghuis D.O., Stevens‐Kroef M., & Hoischen A. (2020). Next generation cytogenetics: comprehensive assessment of 48 leukemia genomes by genome imaging.
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