This chapter describes a polymerase chain reaction (PCR)-based method for producing large quantities of BAC DNA, which aims at maximizing the representation of each bacterial artificial chromosome (BAC). Ligation-mediated PCR is used as a template in a Re-PCR reaction to generate DNA for spotting. Hybridization of fluorescently labeled genomic DNA for array CGH analysis involves distributing 1 g of dextran sulfate over the entire length of a 15-ml tube. While holding the tube horizontally, one needs to squirt in 5 ml of formamide. One needs to carefully aspirate and discard the supernatant, while wiping excess liquid from the tube with a tissue paper. One should be careful not to disturb the pellet. The next step involves placing the slides in the Stratalinker with arrays facing up. The arrays should be given a fixed amount of energy instead of the other available options the Stratalinker might have, such as autocrosslink or time. Overcross-linking the slide might result in a decrease in fluorescent hybridization signal.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)