Genomic DNA (gDNA) from young leaves of all selected species were isolated with the C-TAB method (Doyle and Doyle 1990 ). DNA from all plants were obtained at the same stage. After the extraction of DNA, samples were labeled by nick translation with tetramethylrhodamine-5-dUTP (Sigma-Aldrich). The ribosomal sequences 35S rDNA and 5S rDNA were labeled with digoxigenin-11-dUTP by nick translation and with tetramethyl-rhodamine-5-dUTP (Sigma-Aldrich) by polymerase chain reaction (PCR), respectively (Kwiatek et al. 2016b (link)).
Tetramethylrhodamine 5 dutp
Manufactured by Merck Group
Sourced in United States
Tetramethylrhodamine-5-dUTP is a fluorescent nucleotide analog used in various molecular biology applications. It serves as a labeling agent, enabling the detection and visualization of nucleic acid sequences.
Automatically generated - may contain errors
Lab products found in correlation
Digoxigenin 11 dutp, by Merck Group (3 mentions)
Nick translation, by Merck Group (2 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Atto 647, by Jena Biosciences (1 mentions)
Nick translation kit, by Merck Group (1 mentions)
Dm5500b microscope, by Hamamatsu Photonics (1 mentions)
Orca flash4.0 camera, by Leica (1 mentions)
Las x software v2, by Leica (1 mentions)
6 protocols using tetramethylrhodamine 5 dutp
1
Genomic DNA Extraction and Labeling
2
BAC Sequencing and Mapping in Lupinus angustifolius
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BAC clones were originated from the genomic BAC library constructed by Kasprzak et al. [25 (link)]. The BACs used for assignment of linkage groups to chromosome of L. angustifolius [23 (link),24 (link)] and other available in NCBI database are listed in Table 2 (Results section). BAC DNA was isolated using standard miniprep kits (QIAprep Spin; Qiagen, Hilden, Germany). BAC DNA sequencing was carried out using the long-read sequencing (PacBio), as described by Susek et al. [17 (link)], or by the next-generation sequencing (NGS) approach (Illumina) [24 (link)]. Whole BAC sequences (WBS) were imported into Geneious 8.1.6. (Biomatters, Ltd., Auckland, New Zealand) and aligned to the L. angustifolius pseudomolecules and/or scaffolds of Hane et al. [20 (link)]. The BAC-end sequences (BES) were also used when the WBS were not available. For generation of the fluorescence in situ hybridization (FISH) probe, BAC DNA was labeled by nick translation (Sigma-Aldrich, St. Louis, MI, USA), either with digoxygenin-11-dUTP (Sigma-Aldrich), or with tetramethylrhodamine-5-dUTP (Sigma-Aldrich).
3
Genomic DNA Labeling for Comparative Analysis
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Purified DNA of B. nigra cv UP, extracted using the DNeasy plant mini kit (Qiagen), was used for preparing the probe. DNA was sheared in an autoclave for 2 min and then allowed to cool slowly for reannealing. Sheared DNA of the desired fragment size (500–1000 bp) was labeled with Tetramethyl-Rhodamine-5-dUTP (Sigma Aldrich) dye using a nick-translation kit. The DNA of B. napus (200–500 bp) was autoclaved and was used as a blocker at 200 times the probe concentration to prevent nonspecific intergenomic cross-hybridization.
4
Fluorescent Labeling of Forage Grass Genome
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Clone 395, derived from the library representing the most frequently present sequences in the F. pratensis genome, was labeled by nick translation with fluorochrome Atto647 (Jena BioScience) (Majka et al. 2017b (link)). The ribosomal sequence 35S rDNA was labeled with digoxigenin-11-dUTP by nick translation. While 5S rDNA and the Arabidopsis thaliana L. telomere repeats (TTTAGGG)n were labeled by polymerase chain reaction (PCR) with tetramethyl-rhodamine-5-dUTP (Sigma). The total genomic DNA of L. perenne was used as a probe for genomic in situ hybridization and was labeled with digoxigenin-11-dUTP using nick translation kit according to manufacturer instruction (Sigma).
5
BAC Library Clones for Lupinus Genome
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Single copy BAC clones from the L. angustifolius nuclear genome BAC library [33 (link)] identified as Lang06-specific in the previous study [17 (link)] were used. Due to the dispersed mapping pattern of 067H16 BAC clone in wild lupins, one additional probe (059F07) specific to Lang06 [34 (link)] was used instead. Moreover, BAC clone 127N17 was also not included in FISH because of its overlapping sequence with the BAC 051D03. The complete list of used BAC clones and their alignment to pseudochromosomes is included in the Supplementary Materials (Supplementary Table S1) . DNA isolation from BAC clones was performed using miniprep kits (QIAprep Spin; Qiagen, Hilden, Germany). BAC DNA thus obtained was labeled by nick-translation (Sigma–Aldrich, St. Louis, MI, USA), using either digoxigenin-11-dUTP (Sigma–Aldrich) or tetramethylrhodamine-5-dUTP (Sigma–Aldrich). BAC clone DNA isolation and labeling were done as described by Susek et al. [35 (link)]. To obtain information on localization of BAC clones in the genome assembly, nucleotide sequences of inserts were downloaded from the NCBI database (accession numbers provided in Table S1 ) and aligned to the L. angustifolius pseudomolecules and/or scaffolds [18 (link)] using Basic Local Alignment Search Tool (BLAST) implemented in Geneious 9.1.8 program (Biomatters, Ltd., Auckland, New Zealand).
6
GISH for H. chilense Genome Characterization
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Chromosome preparation and genome in situ hybridization (GISH) were carried out as described previously (Rey et al. 2018 (link)). H. chilense genomic DNA was directly labelled with tetramethyl-rhodamine-5-dUTP (Sigma) by nick translation. Images were taken using a Leica DM5500B microscope equipped with a Hamamatsu ORCA-FLASH4.0 camera and controlled by Leica LAS X software v2.0.
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