粘粒

Preparation of Cosmid DNA

• Preparation of Cosmid DNA from 50 ml Cultures (Donis Keller Lab) Cosmid 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 of cosmid DNA. This protocol is particularly helpful for obtaining higher yields of the HTY cosmids.    

   

• Fingerprint Analysis of Cosmid DNA (GSC, WUSM)   

   

• Cosmid 96 well mini-Preparation (GSC, WUSM)   

   

• Fosmid/BAC/PAC/cosmid/P1 Preparation (GSC, WUSM)   

   

• Cosmid Preparation (GSC, WUSM)   

   

• Alkaline Lysis Minipreps of Plasmid or Cosmid DNA (Donis Keller Lab) Plasmid 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.   

   

• Large Scale Plasmid, Cosmid, BAC, PAC, and Fosmid DNA Isolation by a Cleared Lysate Method Followed by Double Acetate Precipitation (Roe Lab)   

   

• CsCl/EtBr Cosmid Preparation (Stanford) The initial step in the sequencing procedure is to isolate the cosmid from the original E. coli that was provided by the Olson lab of Washington University, St. Louis. The cosmid preparation is a series of procedures whereby the cosmid DNA is amplified, isolated, and purified from the E coli host. 

   

・         Cosmid Miniprepl   

・         Laurie's Cosmid Preparation   

・         Plasmid or Cosmid DNA Miniprep (Chun-Ming Liu)

Cosmid Packing and Transformation

• Transformation of Plasmids/Cosmids into E. coli (Donis Keller Lab) 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.   

   

• Cosmid packaging protocol     

   

• Sequencing off Cosmid, BAC, PAC, and Fosmid Templates with ABI Big Dye Terminators (Roe Lab)   

   

• Cosmid Cloning: Cell preparation, DNA packaging, and Cell Transfection (NWFSC)

   

Cosmid DNA Sequencing

• Cosmid Direct Sequencing (GSC, WUSM)