Preparation of slides and hybridization using bacterial artificial chromosome—fluorescent in situ hybridization (BAC-FISH) was carried as described in [53 (link)–55 (link)]. A B. distachyon BAC clone, ABR1-63-E6, that hybridizes all chromosomes of B. distachyon, but not those of B. stacei was labelled by random priming with biotin-14-dUTP (Invitrogen, Life Technologies) [56 (link)]. A ribosomal DNA probe, pTa 71,[57 (link)] which contains a 9-kb EcoRI fragment of rDNA repeat unit (18S-5.8S-26S genes and spacers) isolated from Triticum aestivum was labelled with Alexa-488 dUTP by random priming. Biotinylated probe was immunodetected using Texas Red avidin DCS (Vector Laboratories) and the signal was amplified with biotinylated anti-avidin D (Vector Laboratories). The chromosomes were mounted and counterstained in Vectashield (Vector Laboratories) containing 2.5 μg/mL 4’,6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured using a CoolSnap HQ camera (Photometrics, Tucson, Ariz) on an Axioplan 2 microscope (Zeiss, Oberkochen, Germany) and analysed using MetaVueTM (Universal Imaging Corporation,Downington, PA).
Biotin 14 dutp
Manufactured by Thermo Fisher Scientific
Sourced in United States
Biotin-14-dUTP is a biotinylated nucleotide analog that can be incorporated into DNA during in vitro synthesis or labeling. It functions as a convenient label for detection and analysis of DNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Texas red avidin dcs, by Vector Laboratories (3 mentions)
Biotinylated anti avidin d, by Vector Laboratories (3 mentions)
Axioplan 2 microscope, by Zeiss (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Metavue, by Universal Imaging (2 mentions)
Alexa 488 5 dutp, by Thermo Fisher Scientific (2 mentions)
Volocity, by PerkinElmer (1 mentions)
Dmrxa2 fluorescence microscope, by Leica (1 mentions)
Bx61 fluorescence microscope, by Olympus (1 mentions)
6 protocols using biotin 14 dutp
1
Chromosome Identification via BAC-FISH
2
Chromosome Preparation and In Situ Hybridization
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Chromosome preparations and in situ hybridization were performed as described in D’Hont et al. (2000 ) with modifications. BAC clones (Table S3 ) from accession DH‐Pahang (D’Hont et al., 2012 ; http://banana‐genome.cirad.fr ) were labeled by random priming with biotin‐14‐dUTP (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) or Alexa 488‐5‐dUTP (Life Technologies; Thermo Fisher Scientific). Chromosome preparations were incubated in RNAse A (100 ng µl–1) and pepsin (100 mg ml–1) in 0.01 m HCl and fixed with paraformaldehyde (4%). Biotinylated probes were immunodetected by Texas Red avidin DCS (Vector Laboratories, Burlingame, CA, USA) and the signal was amplified with biotinylated antiavidin D (Vector Laboratories). Fluorescence images were captured using a cooled high‐resolution black and white CCD camera (ORCA; Hamamatsu, Hamamatsu City, Japan) fitted on a DMRXA2 fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and analysed using volocity (Perkin Elmer, Waltham, MA, USA).
3
Meiotic Behavior and Chromosome Identification in Brassica napus Hybrids
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Young floral buds were harvested from either AA or AAC F1 hybrids in order to characterize their meiotic behavior from at least 20 Pollen Mother Cells (PMCs) at Metaphase I of meiosis following the protocol of Suay et al. [53 (link)]. BAC-FISH experiments were performed for the AAC F1 hybrids using the B. oleracea BAC clone Bob014O06 [105 (link)] and the B. rapa BAC clone KBrH033J07 [106 ] that were labelled by random priming with Alexa 488-5-dUTP and biotin-14-dUTP (Invitrogen, life technologies), respectively. The BAC KBrH033J07 hybridizes to A05 and C04 chromosome pairs in B. napus whereas the BAC Bob014O06 was used as “genomic in situ hybridization (GISH)-like” to distinguish specifically all C chromosomes in B. napus.
4
Visualizing Insect Telomeres by FISH
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In species with supposed sex chromosome multivalents, we performed FISH with a telomeric probe on slides previously used for CGH to visualize chromosome ends. The insect telomeric probe (TTAGG)n [47 (link)] was generated by non-template PCR [48 (link)] as described in the work of [49 (link)] and labeled with biotin-14-dUTP (Invitrogen) by nick translation (see above) with 50 min incubation. Genomic probes were removed from slides as described in the work of [50 (link)]. The telomeric probe was then hybridized on slides followed by multiple signal amplification steps, using streptavidin-Cy3 conjugated antibodies (Jackson ImmunoRes. Labs., Inc., West Grove, PA, USA) and biotinylated antistreptavidin antibodies (Vector Labs., Inc., Burlingame, CA, USA) according to the protocol described in the work of [49 (link)]. Finally, the slides were counterstained with 0.5 µg/mL DAPI in DABCO antifade.
5
FISH Localization of 18S rDNA in Insects
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FISH was performed according to Pinkel et al. [39 ], with modifications of Cabral-de-Mello et al. [40 (link)]: the 18S rDNA probe obtained from M. v-luteum was used in all insects analyzed. The DNA fragments were labeled with biotin-14-dUTP (Invitrogen) by PCR and the products visualized by 1% agarose gel electrophoresis to verify the amplification of the sequences. FISH signals were detected using alexa-flu-488 (Life Technologies) and the preparations were stained with 4 ', 6-diamidine-2'-phenylindole dihydrochloride (DAPI) and then assembled using Vecta shield (Vector). The preparations were observed using an Olympus BX61 Fluorescence microscope with DP70 refrigerated digital camera. Images were merged and optimized for brightness and contrast using Adobe Photoshop CS2 software.
6
Chromosome-based BAC Localization in Banana
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Chromosome preparations were performed as described in D’Hont et al. (2000) . Seven BAC clones (MAMB_34N11, MAMB_17B03, MAMB_51M04, MAMH_47D06, MAMB_01M16, MAMB_51J24, and MAMH_66D03) from both sides of the breakpoints were selected from a BamH1 and HindIII BAC libraries of accession DH-Pahang (D’Hont et al. 2012 (link); http://banana-genome.cirad.fr/ ; last accessed May 29, 2017). BAC clones were labeled by random priming with biotin-14-dUTP (Invitrogen, Life Technologies) or Alexa 488-5-dUTP (Invitrogen, Life Technologies). In situ hybridization was performed as described in D’Hont et al. (1996) (link) with the following modifications. Chromosome preparations were incubated in RNAse A (100 ng/µl), pepsin (100 mg/ml) in 0.01M HCl and fixed with paraformaldehyde (4%). Biotinylated probes were immunodetected by Texas Red avidin DCS (Vector Laboratories) and the signal was amplified with biotinylated antiavidin D (Vector Laboratories). Fluorescence images were captured using a CoolSnap HQ camera (Photometrics, Tucson, Ariz) via an Axioplan 2 microscope (Zeiss, Oberkochen, Germany) and analyzed using MetaVueTM (Universal Imaging Corporation, Downington, PA).
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