Construct cosmid librariesby isolating large genomic DNA fragments from Monascus ruber

Abstract:

Abstract: [Objective] To isolate large genomic DNA fragments from Monascus ruber for the construction of cosmid libraries. [Methods] Modified phenol-chloroform method was used to isolate genomic DNA. The isolated genomic DNA was digested by Sau3AI to 40kb fragments on average. Then, the fragments were packaged by Stratagene’s Gigapack III XL packaging extract. A pair of degenerate primers were used to amplify a fragment of PKS (polyketide synthase) gene from this cosmid library. [Results] The average size of genomic DNA isolated by this method was larger than 48kb, with a concentration of 5μg/μl. The constructed cosmid libraries had 10 folders coverage of the Monascus spp. genome. A cosmid containing the homologue of PKS gene was obtained by PCR screening. [Conclusion] This modified method of isolating large fragments genomic DNA of Monascus ruber was efficient and feasible.