How do you prepare probes for fluorescence in situ hybridization (FISH)?

The construction of probes for FISH usually involved 3 main steps:

• BAC preparation: Bacteria containing the bacterial artificial chromosome (BAC) with complementary sequences to the target DNA are grown in media. When a desired population of bacteria is obtained, these bacteria are lysed and the BACs are isolated.
• BAC fragmentation: BACs are digested with appropriate restriction enzymes to obtain probes of approximately 200 bp.
• Probe labeling: After cleaning up, the fragmented DNA molecules are labeled with fluorophores or haptens, which can be achieved by various means, such as nick translation, random primed labeling and PCR. The degree of labeling may need to be optimized to give an acceptable signal in FISH.