Stock Solution
A stock solution can be defined as a highly concentrated solution, mostly prepared as a 10x concentrated solution, which can be diluted to prepare a working solution. Stock solutions can also be used as a component to prepare complex solutions containing many ingredients. For example, a stock solution of EDTA (0.5 M EDTA solution) can be used to prepare many solutions which contain EDTA e.g., TAE, TBE, Tris-EDTA, Trypsin-EDTA etc.
Preparation of Stock Solution of Ampicillin (50 mg/ml)
Ampicillin is one of the most extensively used antibiotics as a selection marker in molecular biology and bacteriology. Ampicillin is available as Ampicillin anhydrous, Ampicillin trihydrate and Ampicillin sodium salt. Ampicillin sodium salt is often used to prepare stock solution as it has better solubility in water (>50 mg/ml in water). A 50 mg/ml stock solution of ampicillin can be prepared by dissolving 500 mg ampicillin sodium salt in water to a final volume of 10 ml.
Preparation of 2 X YT Medium
The 2 x YT medium, also known as a 2 x TY medium, is a rich medium. It is recommended for the growth and propagation of E. coli infected with the single strand filamentous bacteriophage M13. It can be prepared by dissolving 16 g tryptone, 10 g yeast extract, and 5 g Sodium chloride (NaCl) in water to a final volume of 1000 ml.
Plasmid Isolation
To isolate plasmid from the host bacteria, cells are first lysed that lead to the release of plasmid and in subsequent steps, the plasmid is purified from the lysate. Purification of plasmid from the lysed cells are mostly dependent on the type of lysis method used to release plasmid in solution. For example, alkaline lysis which completely disrupts the bacterial cells leading to the release of cell components including both plasmid DNA and genomic DNA in denatured state relies on selective renaturation of only plasmid DNA in a perfect manner at the purification step. On the other hand, boiling lysis selectively releases only plasmid DNA from the bacterial cells. The purified plasmid can be further purified by a number of methods to obtain high quality plasmids. These methods are centrifugation in gradients of CsCl – ethidium bromide (EtBr), selective precipitation in high salt SDS, extraction with Phenol-chloroform, and hydroxylapatite and ion-exchange chromatography.