质粒的小量制备

 Standard (alkaline lysis) Mini-Prep (Goldberg Lab) Standard protocol for mini-prep and recipe for solution I, II and III.

• 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.   

・         Plasmid Mini-preps (NWFSC) Diatomaceous earth-based and non-diatomaceous earth-based mini-prep. This is a typical mini-prep until step 9, where in step 9a you would precipitate the template and uses it for Taq Cycle Sequencing with the Dye-Labeled Primers, or in step 9b proceed with the diatomaceous earth purification for Taq Dye-Labeled Terminator Cycle Sequencing Reactions.

・         Miniprep double-stranded DNA isolation (ROE) After collecting the plasmid containing cells by centrifugation, the cell pellet is resuspended in a hypotonic sucrose buffer. The cells are successively incubated with an RNase-lysis buffer, alkaline detergent, and sodium acetate. The lysate is cleared of precipitated proteins and membranes by centrifugation, and the plasmid DNA is recovered from the supernatant by isopropanol precipitation. The DNA is crudely checked for concentration and purity using agarose gel electrophoresis against known standards. A typical yield for this method of DNA isolation is 10-15 ug of plasmid DNA from a 6 ml starting culture.

・         Plsmid DNA Isolation from Bacteria (LaboratoryExperiments.com) Basic procedure for high school students, But very useful if you are new to molecular biology.

・         Boiling Lysis Miniprep (Brent Graveley)

・         Mini Prep by Boiling (Howell Lab)

・         Quick DNA Plasmid Prep. : Boiling 10 minute protocol (Mike A Dyer)  This is a very fast mini-prep protocol which is suitable for sequence analysis and restriction digests. Although the yield is higher, there is considerable chromosomal DNA, RNA and protein contamination.

・         Quick Plasmid DNA Preparation (Mike A Dyer)  This protocol gives very clean plasmid preps for restriction digests and cloning. However, due to the alkaline lysis step, the DNA is often nicked and may not give exceptional sequence data.

・         Alkaline Lysis Miniprep (Brent Graveley)

・         Plasmid Mini and Maxi Prep Methods (Gimila Lab) 

・         Plasmid Miniprep (Erik) Alkaline lysis and ammonium acetate method

・         Mini-Preps by Diatomaceous Earth Method (NWSFC) This cheap and simplified protocol, based on Hansen et al ., 1995, gives high yields of plasmid DNA as well as high purity and is suitable for cloning, PCR, sequencing, site-directed mutagenesis and in vitro transcription, etc. It is an extremely good method for routine application and provides a good alternative when yields tend to be low due to low plasmid copy number for any number of reasons 

・         Plasmid Minipreps (NWFSC) Standard method of miniprep.

・         96-Well Plasmid Minipreparation (MOD Laboratory) Standard method of miniprep.

・         Ctab Mini Plasmid Preparation (PMCI Research)

・         Alkaline Lysis Mini Plasmid Preps (PMCI Research)

・         Plasmid Quickpreps (Kay Schneitz Lab) Nice protoocl for preparing plasmid DNA from a single colony

・         Minipreps quick (Crawford Lab)

・         DNA Plasmid Prep. : Baron Lab long version (Mike A Dyer)  This is a scaled up version of the Baron protocol which has been modified to achieve purity comparable to CsCl preps.

・         Mini/Maxi Plasmid Prep (Crawford Lab) Standard plasmid prep method

・         CTAB_Miniprep (Templenton Lab)

Plasmid Minipreps (Promega)

・         Plasmid Minipreps (Bowtell Lab)

・         Plasmid Isolation using Good old Phenol (Whitehead Institute/MIT)

・         Preparation of Plasmid from Yeast (Hoshi Lab)

・         Plasmid cDNA MiniPrep (Immunology Resource)

・         "Smash and Grab" Recovery of Plasmids (D. Amberg)

・         Recovering Plasmids from Dried Agar plates or Stabs (Life Technologies)

• Toothpick Plasmid Assay (Donis Keller Lab) The toothpick assay is used to identify bacterial colonies containing recombinant plasmids following transformation and to obtain an approximation of the plasmid ｉｎｓｅｒｔ sizes.   
• Preparing Overnight Bacteria Culture (LaboratoryExperiments.com) This is a basic procedure for high school students and useful for those who are new to molecular biology.