a. What is the function of the SDS in the lysis buffer? the NaOH?
b. What does the potassium acetate do in the procedure and why?
Extracted text: Plasmids were originally isolated from simple organisms such as bacteria, algae or yeasts. They are usually much smaller than chromosomes (1 000 to 20 0o0bp, while chromosomes have millions of base pairs). Plasmids are extrachromosomal circular DNA molecules that replicate autonomously, using their own origins of replication (ori) and encode their own RNA s and proteins. They often carry genes encoding resistance to one or more antibiotics, and can confer this resistance to their bacterial hosts. Molecular biologists have adapted plasmids to serve as vectors to carry foreign DNA fragments that have been inserted (cloned) into them. PUC19 is a popular vector which was derived from a naturally occurring E. coli plasmid that was modified for cloning. The important features are its relatively small size (2,686 bp), a 54 bp polylinker with 21 restriction enzyme recognition sites (the Multiple Cloning Site or MCS), and a short segment of DNA containing the promoter region and first part of the coding sequence for the LacZ (B-galactosidase) gene, that allows for "blue-white" screening for cloned inserts. Figure 3-1. Map of the DUC19 plasmid. Glick& Pastemak. lacZ'gene Amp Multiple cloning sequence gene PUC19 lacl gene Origin of replication The miniprep procedure is used to isolate small plasmid DNA from small volume cultures of bacteria while limiting contaminating proteins and genomic DNA. All plasmid purification protocols take advantage of the relatively small size and covalently closed structure of plasmid DNA. Although there is always some
Extracted text: contamination with RNA and variable amounts of E. coli chromosomal DNA, the plasmid quality is acceptable for restriction analysis, sequencing, and subcloning. E. coli cells containing the plasmid pUC19 are grown in selective media (LB + Ampicillin) to late log phase. PUC19 plasmids replicate to a very high copy number (several hundred) so large amounts of DNA can be prepared from relatively small volumes of transformed bacteria. Bacteria containing the plasmid are harvested by centrifugation, and the pellets lysed to release the plasmids. Of the many methods developed to purify plasmid DNA, the alkaline lysis method (Birnboim and Doly, 1979; Ish-Horowicz and Burke, 1981) is the most popular because of its simplicity, reproducibility and low cost. With this method, bacterial pellets are treated with alkaline detergent. SDS is a strongly anionic detergent that at high pH opens the cell wall, disrupts the cell membranes, denatures chromosomal DNA and proteins, and releases plasmid DNA into the supernatant. The alkali disrupts DNA base pairing and causes the linear stretches of sheared chromosomal DNA to denature; however, the strands of the closed circular plasmid DNA are unable to separate from each other because they are topologically intertwined. As.long.as the intensity and duration of the exposure to OH- is not too great, the strands of the plasmid DNA reanneal when conditions are returned to normal pH, and native super helical circular DNA reforms. Prolonged exposure to denaturing conditions, however, can cause irreversible denaturation, and this DNA is resistant to cleavage with restriction enzymes and stains poorly with intercalating dyes such as ethidium bromide or ŞybrSafeTM. Varying amounts of this form can usually be seen in plasmid preparations prepared by alkaline lysis (or boiling). During lysis, detergent solubilized proteins, broken cell walls and membranes, and denatured chromosomal DNA become enmeshed in large complexes coated with dodecyl sulfate (the DS of SDS). These complexes are efficiently precipitated from solution when sodium ions are replaced by potassium ions. After pelleting the debris, the plasmid DNA is precipitated from the supernatant with ethanol and the DNA resuspended in TE. The expected yields from a 5 mL culture are 5-10 ug of plasmid DNA. The procedure can be scaled up many folds (midi-preps and maxi-preps). The DNA is usually stored at -20 °C for long term; however, to avoid repeated freezing and thawing of DNA it should be stored at 4 °C for immediate use. A large number.of kits for plasmid DNA purification are now available from many commercial vendors including Monarch® These kits consist of disposable chromatography columns that are used for absorption then elution of plasmid DNA. Many different matrices are available including glass, diatomaceous earth and anionic resins such as DEAE (diethylaminoethyl); the Purelink™M columns from Monarch® have a silica membrane. All the necessary buffers and columns are provided, but they are relatively expensive and do not perform significantly better than standard reagents used in routine miniprep protocols. The Purelink Quick Plasmid Miniprep KitTM from Monarch® is designed to isolate up to 30 µg of high quality plasmid DNA from E. coli cells in 30 to 45-minutes. The bacterial cells are lysed using an alkaline/SDS procedure. The lysate is then applied to a Spin Column that selectively binds plasmid DNA. Contaminants are removed with two Wash Buffers. The plasmid DNA is then eluted in TE buffer.