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.
Alexa 488 5 dutp
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Alexa Fluor 488-5-dUTP is a fluorescent nucleotide analog that can be incorporated into DNA or RNA for detection and visualization applications. It features the Alexa Fluor 488 fluorescent dye, which has high brightness and photostability.
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2 protocols using alexa 488 5 dutp
1
Meiotic Behavior and Chromosome Identification in Brassica napus Hybrids
2
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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