Yang An1, Jing Yang, Xinxin Xu, Gang Liu. 1. Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. anyang@foxmail.com
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 48 kb, with a concentration of 5 microg/microl. 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.
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 48 kb, with a concentration of 5 microg/microl. 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.