To utilize competent E. coli bacteria to replicate a specific DNA fragment. Three methods are presented transformation of intact plasmids/cosmids ,transformation of ligation mixes and color selectability procedures.
Tuesday,Jul 14,2009 Source: Donis Keller Labhttp://www.genome.ou.edu/big_dyes_cos_bac_pac_fos.html
Tuesday,Jul 14,2009 Source: Roe LabCosmid vectors containing foreign DNA inserts are known to replicate more slowly than small plasmids in culture. One of the reasons is believed to be the capacity of the cosmids to take larger insert DNAs (up to 45 kb) causing an increased DNA replication time. Routine plasmid miniprep protocols using 5 ml overnight cultures yield very little cosmid DNA. The following protocol is a scaled-up version of the 5 ml plasmid miniprep protocol with yields ranging from 5 ug to 10 ug o
Tuesday,Jul 14,2009 Source: Donis Keller Labhttp://www.plant.dlo.nl/projects/hybtech/Liu/pcminiprep.html
Tuesday,Jul 14,2009 Source: Chun-Ming Liuhttp://www.genome.ou.edu/DblAcetateProcV3.html
Tuesday,Jul 14,2009 Source: Roe LabDetailed protocol for preparing cosmid DNA.
Tuesday,Jul 14,2009 Source: DNA Sequencing Facility, Univehttp://www.nwfsc.noaa.gov/announcements/micro-server.html
Tuesday,Jul 14,2009 Source: NWFSCPlasmid or cosmid containing E. coli cells are grown and lysed with alkali. The cell debris and chromosomal DNA is precipitated with SDS and potassium acetate. After pelleting the debris the plasmid/cosmid DNA is precipitated from the supernatant with isopropanol and the DNA resuspended in TE. The expected yields from a 5 ml culture are 5-10 ug for plasmids 2-5 ug for cosmids. The procedure can be scaled up many fold.
Tuesday,Jul 14,2009 Source: Donis Keller Lab
