Total genomic DNA was extracted from fresh leaves of Ae. tauschii (DD), Triticum monococcum (AmAm), Ae. speltoides (BB), S. cereale (RR), and triticale ‘Bogo’ (AABBRR) using GeneMATRIX Plant and Funghi DNA Purification Kit (EURx Ltd.). Genomic DNA from Ae. tauschii and T. monococcum were labeled by nick translation (using NickTranslation Kit, Roche, Mannheim, Germany) with tetramethyl-5-dUTP-rhodamine (Roche) or digoxigenin-11-dUTP (Roche) depending on the visualization concept. Blocking DNA from triticale, Ae. speltoides and S. cereale, was sheared to fragments of 5–10 kb by boiling for 30–45 min and used at a ratio of 1:50 (probe:block). The 5S rDNA and 25S rDNA as well as pSc119.2 and pAs1 probes were obtained as have been described by Kwiatek et al. (2016a (link), b (link)) and labeled with tetramethyl-rhodamine-5-dUTP (Roche), digoxigenin-11-dUTP (Roche), digoxigenin-11-dUTP (Roche), and tetramethyl-rhodamine-5-dUTP (Roche), respectively. Digoxigenin detection was made using anti-digoxigenin-fluorescein antibody (Roche).
Tetramethyl rhodamine 5 dutp
Manufactured by Roche
Sourced in Germany, Switzerland
Tetramethyl-rhodamine-5-dUTP is a fluorescently-labeled nucleotide used in various molecular biology applications. It contains a tetramethyl-rhodamine dye molecule attached to the 5-position of the deoxyuridine triphosphate. This dye-labeled nucleotide can be incorporated into DNA or RNA during enzymatic synthesis, allowing for detection and visualization of the labeled nucleic acid sequences.
Automatically generated - may contain errors
Lab products found in correlation
Digoxigenin 11 dutp, by Roche (11 mentions)
Fluorescein 12 dutp, by Roche (4 mentions)
Nick translation mix, by Roche (3 mentions)
Nick translation kit, by Roche (3 mentions)
Qiaprep spin miniprep kit, by Qiagen (2 mentions)
Fluorescein 12 2 deoxy uridine 5 triphosphate, by Roche (2 mentions)
Genematrix plant and funghi dna purification kit, by EURx (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Atto647n, by Jena Biosciences (2 mentions)
Digoxygenin 11 dutp, by Roche (1 mentions)
Fitc conjugated anti digoxigenin primary antibodies, by Roche (1 mentions)
Cell f software, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Bx60 microscope, by Olympus (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Provis ax70, by Olympus (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Bf100 58 10, by Sutter Instruments (1 mentions)
Tev protease, by GenScript (1 mentions)
Halocarbon oil 700, by Merck Group (1 mentions)
24 protocols using tetramethyl rhodamine 5 dutp
1
Genomic DNA Extraction and Labeling
2
Fluorescent Labeling of Genomic DNA Probes
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All cDNA probes were labeled by nick translation as described previously, with minor modifications (Kato et al. 2004 (link), 2006 (link)). Briefly, all cDNA amplifications were labeled with tetramethyl-rhodamine-5-dUTP (Roche Diagnostics GmbH, Mannheim, Germany Cat. # 11534378910). After the addition of EDTA (500 mM, pH 8.0) to terminate the nick translation reaction, the probes were purified using the Omega DNA Probe Purification Kit (Cat. # D6538-02) following the manufacturer’s recommendations.
The oligonucleotide probes were used for 5S rDNA, 45S rDNA, pAs1, and (AAG)10. The designated oligonucleotides pAs1-1 plus pAs1-2, 5Sg, Oligo-pTa71-2 representing pAs1, 5S and 45S rDNA respectively (Danilova et al. 2012 (link); Tang et al. 2014 (link)). All oligonucleotides were end-labeled using either fluorescein amidite (FAM ; green) or carboxy tetramethyl rhodamine (TAMRA ; red) (Sangon Biotech Co., Ltd., Shanghai, China).
Genomic DNAs ofPseudoroegneria stipifolia Aropyron cristatum
The oligonucleotide probes were used for 5S rDNA, 45S rDNA, pAs1, and (AAG)10. The designated oligonucleotides pAs1-1 plus pAs1-2, 5Sg, Oligo-pTa71-2 representing pAs1, 5S and 45S rDNA respectively (Danilova et al. 2012 (link); Tang et al. 2014 (link)). All oligonucleotides were end-labeled using either fluorescein amidite (
Genomic DNAs of
3
Probing rDNA sequences in plants
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The wheat (T. aestivum var. Chinese spring) clone pTa794 (Gerlach and Dyer 1980 (link)) and A. thaliana 2.3-kb ClaI subclone (Unfried and Gruendler 1990 (link)) were used as 5S rDNA and 25S subunit of 45S rDNA reference sequences, respectively. rDNA probes as well as narrow-leafed lupin BAC clone 072O21 carrying 45S rDNA sequence (Książkiewicz et al. 2013 (link)) were labeled with digoxigenin-11-dUTP and/or tetramethyl-rhodamine-5-dUTP by nick translation (Roche Diagnostics, Basel, Switzerland).
L. angustifolius sequence 120E23_5 (GF110967.1) annotated as 5S rDNA (Książkiewicz et al. 2013 (link)) was labeled with tetramethyl-rhodamine-5-dUTP (Roche Diagnostics, Basel, Switzerland) by PCR according to Hasterok et al. (2004 (link)).
L. angustifolius sequence 120E23_5 (GF110967.1) annotated as 5S rDNA (Książkiewicz et al. 2013 (link)) was labeled with tetramethyl-rhodamine-5-dUTP (Roche Diagnostics, Basel, Switzerland) by PCR according to Hasterok et al. (2004 (link)).
4
BAC-FISH Protocol for Chromosomal Analysis
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DNA was isolated from single Escherichia coli colonies (QIAprep Spin Miniprep Kit; Qiagen) (Farrar and Donnison 2007 (link)), labeled with digoxygenin-11-dUTP and/or tetramethylrhodamine-5-dUTP (Roche Diagnostics) by nick translation, and used as molecular probes for BAC-FISH. In some cases, two or three BAC clones were simultaneously analyzed in various combinations (multi-BAC-FISH). These studies were carried out on mitotic metaphase chromosomes. Cytological preparations were made from root meristematic tissues, as previously described (Lesniewska et al. 2011 (link)). Slide quality was controlled by observation under a phase-contrast microscope (BX41; Olympus). FISH was performed according to the protocol previously adapted for use in L. angustifolius (Lesniewska et al. 2011 (link); Książkiewicz et al. 2013 (link)). Digoxygenated DNA probes were detected with FITC-conjugated antidigoxigenin primary antibodies (Roche Diagnostics). Chromosomes were counterstained with 2 μg/ml 4',6-diamidino-2-phenylindole (DAPI) (Sigma) in Vectashield antifade mounting medium (Vector Laboratories, Burlingame, CA). Preparations were examined under a BX 60 microscope (Olympus) using the Cell_F software (Olympus). The images were captured using a CCD monochromatic camera and superimposed using Micrografx Picture Publisher 8 software (Corel).
5
Metaphase FISH Mapping of Patient Chromosomes
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Metaphase chromosome spreads of the patients were prepared from primary mitotic fibroblasts. BAC clones (RP11-139D07 for the 2N4 and RP11-327M19 for the 2N7 patient) were selected from the Wellcome Trust Sanger Institute Ensembl contigs and obtained from the Resource Center Primary Database of the German Human Genome Project and ResGen (Invitrogen). Genomic BAC DNAs were labeled with Tetramethyl-rhodamine-5-dUTP* (Roche) or 25 nmol Fluorescein-12-dUTP* (Roche) by standard nick-translation and FISH-mapped on metaphase chromosomes. Control BAC clones were chosen for 16q terminal or 2q terminal chromosome areas. Images were generated using the Leica microscope CTR MIC and Software CW4000.
6
Fluorescence In Situ Hybridization of Plant Chromosomes
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Root tip microscope slides were prepared according to previously described protocols [24 (link),34 (link)]. Chromosome slide preparation was performed with the air-dry method.
The selected BAC DNA was extracted using HiSpeed Plasmid Kits, and whole BAC DNA was used for the labeling. A Nick translation kit was used to label insert fragments with tetramethyl-rhodamine-5-dUTP (Roche Diagnostics, Mannheim, Germany).
The polymerase chain reaction (PCR) was used to amplify 45S rDNA fragments, following the method of Sogin [35 ]. Types III and IV tandem repeats of cucumber were amplified following Helm and Hemleben [36 (link)] method.
The FISH was carried out with a modified method for direct labeling according to Tagashira et al. [24 (link)].
FISH signal photomicrographs were taken under an Olympus AX70 “Provis” (Olympus, Tokyo, Japan) microscope equipped with an Olympus DP50 digital camera. The image processing was performed using Photoshop (Adobe, Los Angeles, CA, USA) [37 ], pseudo-colored by increasing the color definition of signals based on differential coloration.
The selected BAC DNA was extracted using HiSpeed Plasmid Kits, and whole BAC DNA was used for the labeling. A Nick translation kit was used to label insert fragments with tetramethyl-rhodamine-5-dUTP (Roche Diagnostics, Mannheim, Germany).
The polymerase chain reaction (PCR) was used to amplify 45S rDNA fragments, following the method of Sogin [35 ]. Types III and IV tandem repeats of cucumber were amplified following Helm and Hemleben [36 (link)] method.
The FISH was carried out with a modified method for direct labeling according to Tagashira et al. [24 (link)].
FISH signal photomicrographs were taken under an Olympus AX70 “Provis” (Olympus, Tokyo, Japan) microscope equipped with an Olympus DP50 digital camera. The image processing was performed using Photoshop (Adobe, Los Angeles, CA, USA) [37 ], pseudo-colored by increasing the color definition of signals based on differential coloration.
7
Investigating Centromere Localization in Trypanosoma brucei
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Trypanosoma brucei procyclics were fixed in 4% paraformaldehyde and 4% acetic acid, air-dried on microscope immunofluorescence slides and dehydrated in serial ethanol baths (50–100%). Probes were labelled with tetramethyl-rhodamine-5-dUTP® (Roche) by using the Nick Translation Mix® (Roche). Slides were then hybridized with a heat-denatured DNA probe under a sealed rubber frame at 94°C for 2 min and then overnight at 37°C. The hybridization solution contained 50% formamide, 10% dextran sulfate, 2X SSPE, 250-mg/ml salmon sperm DNA and 100 ng of labelled double strand DNA probe. After hybridization, parasites were sequentially washed in 50% formamide-2 X SSC at 37°C for 30 min, 2X SSC at 50°C for 10 min, 2X SSC at 60°C for 10 min and 4X SSC at room temperature. Slides were finally mounted in Vectashield (Vector Laboratories) with DAPI and microscopically examined; more than 200 cells per transfected strain were counted. For statistical analysis of centromere localization, data for 200 cells were compared using the chi-square test. After inhibition of TbMlp2 by RNAi, the number of copies of chromosome 1 was determined by using DNA probes targeting the alpha- and beta-tubulin genes (55 (link)).
8
Probes for Chromosome Localization
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Two kinds of probes were used–(i) 5S rDNA probe was generated by the PCR amplification of a 410-bp BamHI sub-clone of the 5S rDNA from the wheat clone pTa794 [63 (link)] and labelled by PCR with tetramethyl-rhodamine-5-dUTP (Roche) using the universal M13 ‘forward’ (5`-CAG GGT TTT CCC AGT CAC GA-3`) and ‘reverse’ (5`-CGG ATA ACA ATT TCA CAC AGG A-3`) sequencing primers. The thermal cycling programme was as follows: 94°C for 1 min, 35 cycles of 94°C for 40 s, 55°C for 40 s and 72°C for 90 s and finally 72°C for 5 min and (ii) 26S rDNA probe, which was used to detect the 35S rDNA loci, was made by the nick translation of a 2.3-kb ClaI sub-clone of the 26S rDNA coding region of Arabidopsis thaliana [64 (link)] with digoxigenin-11-dUTP (Roche). The conditions for this reaction were as follows: 15°C for 95 min and 65°C for 10 min.
9
Germline Microinjection in C. elegans
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Germline injections were performed in young adult worms at 18–24 h post L4 immobilised in 2% agarose pads and covered with Halocarbon oil 700 (Sigma) using a Narishige IM-31 pneumatic microinjector attached to an inverted Olympus IX71 microscope. Needles were made using borosilicate glass filaments with a 1.0 mm O.D. and 0.58 mm I.D. (BF100-58-10, Sutter Instruments) and a micropipette puller P-97 (Intracell). In all experiments except those displayed in Supplementary Fig. 2a , AcTEVTM Protease (Thermo Fisher, Cat. No. 12575) was used in a mix containing 10U/µl TEV protease in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM DTT, 50% (v/v) glycerol, 0.1% (w/v) Triton X-100. For Supplementary Fig. 2a we used TEV protease from GenScript (Cat. No. Z03030-1000) in a final mix containing 2.5 ng/µl TEV protease in 50 mM Tris, 5 mM DTT, 12.5% glycerol, pH 7.5. In indicated experiments 25 pmol of tetramethyl-rhodamine-5-dUTP (Roche) were added to the injection mix to evaluate the efficiency of germline microinjection by the incorporation of labelled nucleotides into the DNA of germ cells. Following microinjection, worms were rescued from the agarose pad with M9 salt buffer and placed in NG plates with Escherichiacoli OP50 for 3h30’ (unless otherwise indicated) before germline dissection.
10
Detecting conserved rDNA sites in Arabidopsis
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To detect evolutionarily conserved 18S-5.8S-26S and 5S rDNA sites, a 2.3 kb ClaI fragment of the 26S rDNA ribosomal gene of A. thaliana (Unfried and Gruendler 1990 (link)) labeled with digoxigenin-11-dUTP (Roche Applied Science) by nick translation and the 5S rDNA coding unit, PCR-labeled with tetramethyl-rhodamine-5-dUTP (Roche Applied Science), were obtained as previously described (Golczyk 2011a (link); Golczyk et al. 2014 (link)) and used as probes.
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