Whole Chromosome Paints (WCPBs) were derived from two Bs of the A. flavicollis female #24985 (WCP24985aB and WCP24985bB), one of Bs of the male #3727 (WCP3727B), one of Bs of females #3980 (WCP3980B), #3977 (WCP3977B) and #26368 (WCP26368B). Probes were generated earlier by metaphase chromosome microdissection and DNA amplification using a GenomePlex Single Cell Whole Genome Amplification Kit (WGA4) (Sigma-Aldrich, Saint Louis, MO, USA) according to the manufacturer’s protocol [41 (link)], labeled with TAMRA-5-dUTP (Roche) and used for 2D- and 3D-FISH.
Tamra 5 dutp
Manufactured by Roche
Sourced in United States
TAMRA-5-dUTP is a fluorescently labeled nucleotide analog used in various molecular biology applications. It serves as a substrate for DNA synthesis, allowing the incorporation of the fluorescent label into DNA sequences.
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Lab products found in correlation
2 protocols using tamra 5 dutp
1
Whole Chromosome Paint Generation from Microdissected Bs
2
3D FISH Hybridization and Visualization
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3D FISH was performed as described (Solovei et al. 2002a (Solovei et al. , 2002b)) . PCP11-AC was labeled with biotin and detected by Streptavidin -Cy5 (Invitrogen, yellow). PCP11-B2pq and WCP9-B8 were labeled with TAMRA-5-dUTP (Roche, red) and Alexa Fluor 488-5-dUTP (Invitrogen, green), respectively. The probes were dissolved in hybridization mixture (50% formamide, 10% dextran sulfate, 2×SSC, 0.01% NP-40), loaded on the slide with pretreated cells, covered with coverslip (24×24 mm), and sealed with rubber cement. The DNA of nucleus and probes was denatured on the hot-block of Thermomixer comfort, Eppendorf at 73°C for 5 min. Hybridization was performed for 2 days in humid boxes at 37°C. Post-hybridization D r a f t 9 washing was performed in 0.1×SSC at 60°C and 4 SSC/0.1% NP-40 solutions at 45°C for 5 min.
Then nuclear DNA was counterstained with DAPI and mounted in Invitrogen Prolong Gold Antifade.
Then nuclear DNA was counterstained with DAPI and mounted in Invitrogen Prolong Gold Antifade.
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