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DNA Fragmentation

DNA fragmentation is a biological process in which the DNA molecule is broken down into smaller fragments.
This process can occur naturally during cellular events like apoptosis or as a result of external factors like ionizing radiation or chemical exposure.
Studying DNA fragmentation is crucial for understanding various genetic disorders, cancer, and other diseases.
PubCompare.ai's AI-driven platform helps researchers optimize their DNA fragmentation research by providing a comprehensive comparison of fragmentation protocols from the literature, preprints, and patents.
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Most cited protocols related to «DNA Fragmentation»

1
Single-Strand DNA Library for Ancient Genomics
DNA libraries for sequencing are normally prepared from double-stranded DNA (Fig. 1). However, for ancient DNA the use of single-stranded DNA may be advantageous as it will double its representation in the library. Furthermore, in a single-stranded DNA library, double-stranded molecules that carry modifications on one strand that prevent their incorporation into double-stranded DNA libraries could still be represented by the unmodified strand. We therefore devised a single-stranded library preparation method wherein the ancient DNA is dephosphorylated, heat denatured, and ligated to a biotinylated adaptor oligonucleotide, which allows its immobilization on streptavidin-coated beads (Fig. 1). A primer hybridized to the adaptor is then used to copy the original strand with a DNA polymerase. Finally, a second adaptor is joined to the copied strand by blunt-end ligation and the library molecules are released from the beads. The entire protocol is devoid of DNA purification steps, which inevitably cause loss of material.
We applied this method to aliquots of the two DNA extracts (as well as side fractions) that were previously generated from the 40 mg of bone that comprised the entire inner part of the phalanx (2 (link), 8 ). Comparisons of these newly generated libraries to the two libraries generated in the previous study (2 (link)) show at least a 6-fold and 22-fold increase in the recovery of library molecules (8 ), which is particularly pronounced for longer molecules (Fig. S4).
In addition to improved sequence yield, the single-strand library protocol reveals new aspects of DNA fragmentation and modification patterns (8 ). Since the ends of both DNA strands are left intact, it reveals that strand breakage occurs preferentially before and after guanine residues (Fig. S6), suggesting that guanine nucleotides are frequently lost from ancient DNA, possibly as the result of depurination. It also reveals that deamination of cytosine residues occurs with almost equal frequencies at both ends of the ancient DNA molecules. Since deamination is hypothesized to be frequent in single-stranded DNA overhangs (9 (link), 10 (link)), this suggests that 5′- and 3′-overhangs occur at similar lengths and frequencies in ancient DNA.
Meyer M., Kircher M., Gansauge M.T., Li H., Racimo F., Mallick S., Schraiber J.G., Jay F., Prüfer K., de Filippo C., Sudmant P.H., Alkan C., Fu Q., Do R., Rohland N., Tandon A., Siebauer M., Green R.E., Bryc K., Briggs A.W., Stenzel U., Dabney J., Shendure J., Kitzman J., Hammer M.F., Shunkov M.V., Derevianko A.P., Patterson N., Andrés A.M., Eichler E.E., Slatkin M., Reich D., Kelso J, & Pääbo S. (2012). A HIGH COVERAGE GENOME SEQUENCE FROM AN ARCHAIC DENISOVAN INDIVIDUAL. Science (New York, N.Y.), 338(6104), 222-226.
Publication 2012
Bones Bones of Fingers Cytosine Deamination DNA DNA, Ancient DNA, Double-Stranded DNA, Single-Stranded DNA-Directed DNA Polymerase DNA Fragmentation DNA Library Guanine Guanine Nucleotides Immobilization Ligation Oligonucleotide Primers Oligonucleotides Streptavidin
2
DNA Fragmentation Using Covaris E210
We used the Covaris E210 AFA instrument (Woburn, MA) (Quail et al. 2008 (link), 2009 ; Fisher et al. 2011 (link)) to shear the DNA into fragments of a desired length. A previous study on automated 454 library preparation (Lennon et al. 2010 (link)) showed that it is possible to use 96-well PCR plates on the E210, and we adapted the method to another plate type and a shorter fragment size (Supplemental Notes, “DNA Fragmentation”). This reduces consumable costs for the shearing step by ∼90-fold compared with the microTUBE plates provided by Covaris.
Rohland N, & Reich D. (2012). Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Research, 22(5), 939-946.
Publication 2012
DNA Fragmentation Quail
3
Screening for Genes Enabling Robust Growth
The screens for genes required for robust growth were conducted essentially as previously described (Gilbert et al., 2014 (link)). Briefly, plasmid libraries were packaged into lentivirus in HEK293T cells (RRID:CVCL_0063) and infected into a previously established polyclonal K562 cell line stably expressing dCas9-KRAB grown in 3L spinner flasks (Bellco, Vineland, NJ). After two days, infected cells were selected with 0.75 μg/mL puromycin (Tocris, Bristol, UK) for two days, allowed to recover for one day, and then cultured at a minimum of 750 × 106 cells in 1.5L standard media (RPMI-1640 with 10% Fetal Bovine Serum and 1x supplemental glutamine, penicillin, and streptomycin) from 'T0' to 'endpoint,' determined by ~10 cell doublings after T0. CRISPRi screen cells were mock-treated with 0.1% DMSO (Sigma-Aldrich, St. Louis, MO) but otherwise left untreated. Screens were performed as independent replicates starting from the infection step. The K562 dCas9-KRAB and SunTag-VP64 cell lines were obtained from (Gilbert et al., 2014 (link)) and had been constructed from K562 cells originally obtained from ATCC (RRID:CVCL_0004). Cytogenetic profiling by array comparative genomic hybridization (not shown) closely matched previous characterizations of the K562 cell line (Naumann et al., 2001 (link)). All cell lines tested negative for mycoplasma contamination (MycoAlert Kit, Lonza, Basel, Switzerland) in regular screenings.
Frozen samples of 250 × 106 cells collected at T0 and endpoint were processed as previously described (Gilbert et al., 2014 (link)), with the substitution of an SbfI restriction digest (SbfI-HF, New England Biolabs, Ipswich, MA) in place of the MfeI digest in the genomic DNA fragmentation and enrichment step. The sgRNA-encoding regions were sequenced on an Illumina HiSeq-4000 using custom primers. Sequencing reads were aligned to the expected library sequences using Bowtie (v1.0.0, [Ben Langmead et al., 2009 (link)]) and read counts were processed using custom Python scripts (available at https://github.com/mhorlbeck/ScreenProcessing) based on previously established shRNA screen analysis pipelines (Bassik et al., 2013 (link); Kampmann et al., 2013 (link)). sgRNAs represented with fewer than 50 sequencing reads in both T0 and Endpoint were excluded from analysis. sgRNA growth phenotypes (γ) were calculated by normalizing sgRNA log2 enrichment from T0 to endpoint samples and normalizing by the number of cell doublings in this time period. CRISPRi v1 screen data from Gilbert et al. (2014) (link) was re-analyzed using this pipeline, and the hCRISPRi/a-v2 5 sgRNA/gene libraries were evaluated by analyzing the sgRNA read counts corresponding to only the 5 sgRNA/gene sublibraries. Gene ontology analysis was conducted using DAVID 6.7 (Huang et al., 2009 (link)) with default search categories and with background lists representing the genes targeted by the CRISPRa v1 or hCRISPRa-v2 libraries where appropriate. For Figure 4B and Figure 4—figure supplement 1D, 'shared hit' genes were 70 genes that scored as strong anti-growth hits (phenotype z-score x –log10 p-value ≤ −10) in both CRISPRa v1 and hCRISPRa-v2.
Horlbeck M.A., Gilbert L.A., Villalta J.E., Adamson B., Pak R.A., Chen Y., Fields A.P., Park C.Y., Corn J.E., Kampmann M, & Weissman J.S. (2016). Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife, 5, e19760.
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Publication 2016
Cell Lines Cells Crossbreeding Cultured Cells Dietary Supplements DNA Fragmentation DNA Library Fetal Bovine Serum Freezing Gene Library Genes Genome Glutamine Infection K562 Cells Lentivirus Mycoplasma Oligonucleotide Primers Penicillins Phenotype Plasmids Puromycin Python Screenings, Genetic Short Hairpin RNA sodium-binding benzofuran isophthalate Streptomycin Sulfoxide, Dimethyl
4
Targeted Sequencing of Disease-Associated Genes
A custom capture array (NimbleGen, Roche) was designed to capture all exons (3382), splice sites and the immediately adjacent intron sequences of 193 genes known to be associated with three main types of hereditary diseases according to GeneReviews (NCBI), including single-gene disorders, malignant arrhythmias and cardiomyopathies, as well as familial malignant neoplasms.
The methods used for DNA target capture, enrichment and elution followed previously described protocols with minor modifications (Roche NimbleGen, Inc.). Briefly, genomic DNA from peripheral blood was extracted from the 110 samples using the QIAamp DNA BloodMiNi Kit (Qiagen, Hilden, Germany), and fragmentation of the DNA into fragments ranging from 200 bp to 300 bp was performed using an ultrasonoscope (Covaris S2, Massachusetts, USA). Next, 1 µg of purified DNA (quantified by NanoDrop) was treated with T4 DNA polymerase, T4 phosphonucleotide kinase and the Klenow fragment of Escherichia coli DNA polymerase to fill 5′ overhangs and remove 3′ overhangs. Terminal A residues were added following a brief incubation with dATP and the Klenow 3′-5′ exo-enzyme following standard Illumina protocols [13] . Adapter oligonucleotides from Illumina (single reads) were ligated to the ends. After the ligation was completed, successful adapter ligation was confirmed by 4-cycle PCR using a high-fidelity polymerase with PCR primers containing a custom-synthesized barcode sequence (8 bp) as a sample index signature. PCR was used to generate a library for further analysis, and the DNA adapter-ligated and indexed fragments from 10 libraries were pooled and hybridized to the capture array for 72 h. After hybridization and washing, the DNA fragments bound to the array were eluted using 300 ml of elution buffer (Qiagen, Valencia, CA, USA) for each array. A gasket (Agilent) was applied and placed on a in-house constrcted thermal elution device for 20 min at 95°C. We repeated this process once, adding 200 ml of elution buffer (Qiagen). After hybridization of the sequencing primer, base incorporation was carried out on Illumina HiSeq2000 Analyzers (following the manufacturer's standard cluster generation and sequencing protocols) for 90 cycles of sequencing per read to generate paired-end reads including 90 bps at each end and 8 bps of the index tag. Image analysis and base calling were performed using the Illumina Pipeline.
Wei X., Ju X., Yi X., Zhu Q., Qu N., Liu T., Chen Y., Jiang H., Yang G., Zhen R., Lan Z., Qi M., Wang J., Yang Y., Chu Y., Li X., Guang Y, & Huang J. (2011). Identification of Sequence Variants in Genetic Disease-Causing Genes Using Targeted Next-Generation Sequencing. PLoS ONE, 6(12), e29500.
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Publication 2011
BLOOD Buffers Cardiac Arrhythmia Cardiomyopathies Crossbreeding DNA-Directed DNA Polymerase DNA Fragmentation DNA Library DNA Polymerase I Enzymes Escherichia coli Exons Genes Genome Hereditary Diseases Introns Ligation Malignant Neoplasms Medical Devices Oligonucleotide Primers Oligonucleotides Phosphotransferases
5
Comprehensive Molecular Characterization of ccRCC
DNA samples from ccRCC patients tumour and matching normal were all obtained under local IRB and LREC approvals for this study and processed as previously described2 (link). DNA fragmentation, library preparation and solution phase hybrid capture were according to manufacturer instructions (Agilent Technologies, US) and modified from previously published protocols5 . Capillary-based Sanger sequencing for confirmations and PBRM1 followup were done as previously described2 (link) with manual inspection of all sequencing traces. mRNA was extracted from snap-frozen mouse pancreatic lesions and subjected to RT-PCR using a nested PCR approach utilising primers of mouse Pbrm1 exon 23/24 and the Carp-β-Actin Splice acceptor sequence of the T2Onc transposon cassette. Resulting bands were gel-purified and subjected to capillary-based Sanger sequencing. PBRM1 or scrambled control siRNAs (Santa Cruz, CA) were transfected into ccRCC cell lines using Lipofectamine 2000 (Invitrogen, CA) according to the manufacturer's conditions. Real-time PCR and western blotting were all done utilising standard protocols essentially as described1 (link). Expression analyses were carried out as previously described2 (link).
Varela I., Tarpey P., Raine K., Huang D., Ong C.K., Stephens P., Davies H., Jones D., Lin M.L., Teague J., Bignell G., Butler A., Cho J., Dalgliesh G.L., Galappaththige D., Greenman C., Hardy C., Jia M., Latimer C., Lau K.W., Marshall J., McLaren S., Menzies A., Mudie L., Stebbings L., Largaespada D.A., Wessels L.F., Richard S., Kahnoski R.J., Anema J., Tuveson D.A., Perez-Mancera P.A., Mustonen V., Fischer A., Adams D.J., Rust A., Chan-on W., Subimerb C., Dykema K., Furge K., Campbell P.J., Teh B.T., Stratton M.R, & Futreal P.A. (2011). Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature, 469(7331), 539-542.
Publication 2010
Actins Capillaries Carps Cell Lines DNA, Neoplasm DNA Fragmentation DNA Library Exons Freezing Hybrids Jumping Genes lipofectamine 2000 Mus Nested Polymerase Chain Reaction Oligonucleotide Primers Patients PBRM1 protein, human Real-Time Polymerase Chain Reaction Reverse Transcriptase Polymerase Chain Reaction RNA, Messenger RNA, Small Interfering

Most recents protocols related to «DNA Fragmentation»

1
DNA Fragmentation for Sequencing
Not available on PMC !

Example 3

1 μL of genomic DNA was processed using a NEBNext dsDNA Fragmentase kit (New England Biolabs) by following the manufacturer's protocol. Incubation time was extended to 45 minutes at 37° C. The fragmentation reaction was stopped by adding 5 μL of 0.5M EDTA pH 8.0, and was purified by adding 2× volumes of Ampure XP beads (Beckman Coulter, A63881) according to the manufacturer's protocol. Fragmented DNA was analyzed on a Bioanalyzer with a High Sensitivity DNA kit (Agilent). The size range of fragmented DNA was typically from about 100 bp to about 200 bp with a peak of about 150 bp.

US11859246B2. Methods and compositions for enrichment of amplification products (2024-01-02). ACCURAGEN HOLDINGS LIMITED [KY]. Inventors: Li Weng [US], Shengrong Lin [US], Lin Fung Tang [US].
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Patent 2024
BP 100 DNA, Double-Stranded DNA Fragmentation Edetic Acid Genome Genomic Library Hypersensitivity
2
Apoptosis Measurement by TUNEL Assay
Fragmentation of intracellular DNA is a late marker of apoptosis and was measured by TUNEL assay, using the APO-DIRECT Kit (556381; BD Biosciences). Briefly, tumor cells treated with rituximab and PBS were fixed with 1% paraformaldehyde, washed, and stored in ice-cold 70% ethanol for at least 24 h before staining with 50 µl of DNA labeling solution, prepared as per the manufacturer’s instructions. After incubation for about 1 h with the DNA labeling solution, the cells were washed and analyzed by flow cytometry.
Saikumar Lakshmi P., Oduor C.I., Forconi C.S., M’Bana V., Bly C., Gerstein R.M., Otieno J.A., Ong’echa J.M., Münz C., Luftig M.A., Brehm M.A., Bailey J.A, & Moormann A.M. (2023). Endemic Burkitt lymphoma avatar mouse models for exploring inter-patient tumor variation and testing targeted therapies. Life Science Alliance, 6(5), e202101355.
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Publication 2023
Apoptosis Biological Assay Cells Cold Temperature DNA Fragmentation Ethanol Flow Cytometry In Situ Nick-End Labeling Neoplasms paraform Protoplasm Rituximab
3
DNA Fragmentation Detection by Fluorometric TUNEL
The DeadEndTM Fluorometric TUNEL System kit (Promega, USA) was used according to the manufacturer’s protocol to detect DNA fragmentation.
Briefly, the samples were fixed in buffered formaldehyde, washed twice in PBS, treated with 0.2% Triton X-100 in PBS, and incubated in an equilibration buffer.
Then, they were incubated in the reaction mix prepared according to the manufacturer’s instructions. The reaction was stopped by adding 2× saline-sodium citrate buffer and was
counterstained with Hoechst (0.1 mg/mL). The slides were evaluated using a fluorescent microscope.
Kermani T., Hosseini S.F., Talaei-Khozani T, & Aliabadi E. (2023). Effect of Pre-Incubation of Cryopreserved Sperm with either Kisspeptin or Glutathione to Mitigate Freeze-Thaw Damage. Iranian Journal of Medical Sciences, 48(2), 198-208.
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Publication 2023
Buffers DNA Fragmentation Fluorometry Formaldehyde In Situ Nick-End Labeling Microscopy Promega Saline Solution Sodium Citrate Triton X-100
4
Metagenomic Next-Generation Sequencing
The intraoperatively obtained tissue was sliced into pieces before being shipped to the laboratory for mNGS. The initial step was to isolate total tissue DNA using the TIANamp Micro DNA Kit (DP316, TIANGEN BIOTECH). After that, Qubit 2.0 (Invitrogen, USA) was used to quantify DNA content, and 200 ng was used in the following step. The second step was to create and identify a DNA library. Initially, DNA fragmentation, end-repair adaption, dA-tailing addition, adapter ligation and PCR amplification were used to create a DNA library. The Agilent 2100 Bioanalyzer (Agilent Technologies, Canada) and qPCR were used to identify the DNA library. To manufacture DNA nanospheres for NGS, the library was cyclized to form a single-stranded ring structure and then rolled to replicate.
After loading on the chip, samples were sequenced at 20 M 50 bp using the BGISEQ-50 platform (BGI Genomics Co., Ltd.). Low quality, adaptor contamination, repeat sequence number, and short (<35 bp) reads were eliminated to obtain high-quality sequencing data. Human reference genome (hg19)-mapped human host sequences were afterward computationally subtracted using the Burrows-Wheeler alignment technique. Meanwhile, the remaining data were classified using simultaneous alignment against four NCBI Microbial Genome Databases (ftp://ftp.ncbi.nlm.nih.gov/genomes/), including bacteria, viruses, fungi, and parasites. They covered 6350 bacterial genomes/scaffolds, 1798 whole genome sequences, 1064 human infection-associated fungi, and 234 human disease-related parasites.
Yu Y., Wang S., Dong G, & Niu Y. (2023). Diagnostic Performance of Metagenomic Next⁃Generation Sequencing in the Diagnosis of Prosthetic Joint Infection Using Tissue Specimens. Infection and Drug Resistance, 16, 1193-1201.
Publication 2023
Acclimatization Bacteria DNA, A-Form DNA Chips DNA Fragmentation DNA Library Fungi Genome Genome, Bacterial Genome, Human Genome, Microbial Homo sapiens Ligation Mycoses Parasites Repetitive Region Tissues Virus
5
Comprehensive Plastome Analysis of Alismatidae
A total of 38 species representing all 12 accepted families in Alismatidae were incorporated into this study. These included 1) 13 species belonging to five families newly sequenced in this study; 2) 21 plastomes from eight families downloaded from GenBank, 3) the sequenced data of the remaining four species, Amphibolis antarctica (SRR19106495), Najas marina (ERR5529706), Posidonia australis (SRR19106496) and Zannichellia palustris (ERR5554861), retrieved from the SRA database (Table S1). Three additional plastomes from Araceae, Tofieldiaceae, and Acoraceae were included as outgroups. The field sampling followed the ethics and legality of the local government and was permitted by the government.
Total genomic DNA extraction, DNA fragmentation and preparation of sequencing libraries of all 13 newly sampled species followed the description by Li et al. [31 (link)]. Genome skimming per sample was conducted on the Illumina HiSeq 2000 platform in Novogene (Tianjin, China), with 150 bp paired-end reads. Newly sequenced reads of 13 species and four SRA sequenced reads were filtered with Fastp v.0.20.1 [45 (link)] using default settings. Complete plastomes were de novo assembled using the software GetOrganelle v.1.7.1 [46 (link)] with default parameters. Plastid Genome Annotator (PGA [47 (link)]) and the online tool Geseq [48 (link)] were utilized for the annotation of assembled plastomes. The annotations for protein-coding genes (PCGs) were checked and adjusted manually according to published plastomes of Alismatales. All newly generated plastomes were deposited in the China National GeneBank DataBase (CNGBdb, https://db.cngb.org/; Table S1).
Li Z.Z., Lehtonen S, & Chen J.M. (2023). The dynamic history of plastome structure across aquatic subclass Alismatidae. BMC Plant Biology, 23, 125.
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Publication 2023
Acoraceae Alismatales Araceae DNA Fragmentation Genes Genome Genome, Plastid Naja Posidonia Protein Annotation

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In situ cell death detection kit by Roche
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The In Situ Cell Death Detection Kit is a laboratory product designed for the detection of programmed cell death, or apoptosis, in cell samples. The kit utilizes a terminal deoxynucleotidyl transferase (TdT) to label DNA strand breaks, allowing for the visualization and quantification of cell death. The core function of this product is to provide researchers with a tool to study and analyze cell death processes.
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Facscalibur by BD
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Cell death detection elisaplus kit by Roche
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The Cell Death Detection ELISAPLUS kit is a quantitative in vitro assay used for the detection and quantification of cytoplasmic histone-associated DNA fragments, which are indicative of apoptosis. The kit provides a simple, fast, and reliable method for the measurement of cell death.
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Deadend fluorometric tunel system by Promega
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The DeadEnd Fluorometric TUNEL System is a laboratory equipment product that detects and quantifies apoptosis, or programmed cell death, in cells. The system utilizes terminal deoxynucleotidyl transferase (TdT) to label DNA strand breaks, which are characteristic of apoptotic cells. The labeled DNA is then detected using fluorescent dyes, allowing for the visualization and quantification of apoptotic cells.
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Cell death detection elisa kit by Roche
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The Cell Death Detection ELISA kit is a quantitative in vitro assay for the detection and quantification of DNA fragmentation, a hallmark of apoptosis. The kit uses an enzyme-linked immunosorbent assay (ELISA) technique to detect and measure cytoplasmic histone-associated DNA fragments.
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The Olympus Fluorescence Microscope is an optical microscope that uses fluorescence to visualize and analyze samples. It illuminates the specimen with light of a specific wavelength, causing fluorescent molecules within the sample to emit light at a different wavelength, which is then detected and displayed.
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The ApoAlert DNA Fragmentation Assay Kit is a laboratory tool that detects and measures DNA fragmentation, a hallmark of apoptosis or programmed cell death. The kit provides a convenient and sensitive method for quantifying DNA fragmentation using a colorimetric approach.
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The FragEL DNA Fragmentation Detection Kit is a laboratory equipment product used to detect and analyze DNA fragmentation. It provides a method for the identification and quantification of apoptotic cells, which is a key cellular process. The kit includes the necessary reagents and materials to perform this analysis.

More about "DNA Fragmentation"

DNA Fragmentation, Apoptosis, Cell Death, TUNEL Assay, Cell Death Detection ELISA, FACSCalibur, Agilent 2100 Bioanalyzer, FragEL DNA Fragmentation Detection Kit, ApoAlert DNA Fragmentation Assay Kit, Fluorescence Microscopy, Propidium Iodide, Genetic Disorders, Cancer