Plasmid Isolation and Identfication

C Plasmid Isolation Introduction to alkaline lysis At times, it is desirable to isolate the plasmid DNA away from the chromosomal DNA, especially when trying to mass a new recombinant plasmid There are a few qualities of the E. coli chromosomal DNA, which help in the separation from plasmid DNA. For example, the chromosomal DNA is attached to the bacterial en membrane However, during isolation of plasmid DNA, care must be taken to ensure that the chromosomal DNA is not sheared into small pieces as this will lead to contamination of the plasmid DNA with the sheared chromosomal DNA Isolating plasmid DNA One of the most common and well-established techniques for isolating plasmid DNA is the alkaline lysis procedure. This is sometimes referred to as a mini-, midi-, or maxi-prep depending on now many cells are used (1) Collection of cells A single colony off of a plate is "picked" and placed into a fresh tube of liquid media plus the selective antibiotic (ampicillin) for our experiment Each cell in the overnight culture should contain and be replicating the same plasmid as the original copy If there is no growth in the overnight culture, the colony may not have contained a plasmid conferring resistance to ampicillin (2) Resuspension and lysis After collection, E. coli are resuspended in a resuspension butter that contains Tris, EDTA, Glucose, and RNase A Tris is the buffering reagent. Glucose helps maintain osmotic balance so that cells do not lyse prematurely. The EDTA helps to weaken the cell membrane and the RNaseA will degrade RNA into small polynucleotides once cells are lysed After resuspension, a lysis buffer is added which contains SDS and NaOH. SDS lyses the cells as well as denatures proteins. NaOH, an alkaline reagent from which the procedure gets its name, denatures proteins and DNA The NaOH disrupts the hydrogen bonds that hold together the two DNA strands of a double helix. By doing so, double-stranded DNA (dsDNA) is converted to single-stranded DNA (ssDNA) (3) Neutralization In the next step, an acidic solution (typically potassium acetate) is added that will neutralize the NaOH. This allows the DNA to renature, or go from ssDNA to dsDNA However, due to the large size of the chromosomal DNA, it does not easily renature properly, and instead parts of the DNA remain single stranded After this step, the plasmid DNA remains in solution, but the solution also contains a high level of salts, and some contaminating cellular proteins (4) Affinity based chromatography An alternate method to the affinity purification is a simple phenol-chloroform extraction. This technique is similar to the phase separation done using chloroform in the genomic DNA purification The phenol in the phenol-chloroform mixture helps in the purification as it is easier to get the initial mixing of phenol-chloroform with the aqueous DNA solution that needs to be purified However, this necessitates a second phase separation step using chloroform alone in order to extract the contaminating phenol from the aqueous phase