6-FAM

6-FAM (6-Carboxyfluorescein) is an isomer of carboxyfluorescein and is mainly used for sequencing and labeling of nucleic acids. In Vitro: In cell experiments, we use 6-Carboxyfluorescein (6-FAM) labeled oligonucleotide probes, mainly through fluorescence in situ hybridization (FISH) technology^[1]
(1) Sample preparation: Prepare chromosomal samples from chicken embryo cells, including steps like colchicine treatment, hypotonic swelling, and methanol-acetic acid fixation.
(2) Pre-treatment: The chromosomes are treated with RNase A, pepsin, and formaldehyde.
(3) Hybridization: The 6-FAM labeled oligonucleotide is dissolved in a hybridization buffer containing E. coli tRNA (50% formamide, 2×SSC, 10% dextran sulfate) at a concentration of 20 ng/μl. After DNA and probes are denatured together, hybridization is done overnight at room temperature in a humidity chamber.
(4) Washing and counterstaining: The slides post-hybridization are washed three times in 2×SSC, then dehydrated through a series of 70%-96% ethanol, air-dried, and mounted with an anti-fade reagent containing DAPI.
(5) Observation and analysis: Use a Leica DM4000B fluorescence microscope equipped with a monochrome CCD camera DFC350FX and appropriate filters to observe the samples, and capture and process the dye images using QFISH software.