Preparation of Reagents required for Plasmid Isolation
To prepare the reagents required for the plasmid isolation.
| NAME | STOCK CONC. | WORKING CONC. |
| Tris(pH 7.5) | 0.7M | 10mM |
| EDTA(pH 8) | 0.5M | 1mM |
| NaOH | 1N | 0.1N |
| SDS | 1.00% | 0.50% |
| Sodium Acetate(pH5.2) | 5M | 3M |
| Ampicillin | Liquid:100ug/ml Solid:50ug/ml | Liquid:200ul Solid:50ul |
| Ethanol | 100.00% | 70%&100% |
...
PRINCIPLE:Functions of the reagents:
01. Tris: Tris interacts with the lipopolysaccharides present on the outter membrane which helps to permeablize and also acts as a buffering reagent.
02. EDTA: It chelates the divalent cations of DNases and hence keeps the DNA in the solution. It binds to Ca2+ and prevents joining of cadherins between cells, preventing clumping of cells grow in liquid suspension.
03. NaOH: It acts by loosening the rigid structures of a cell wall or a membrane, thereby releasing the DNA. Na+ ions block the negative charge of DNA by forming an ionic bond with the negatively charged phosphates on the DNA, neutralizing the negative charges and allowing the DNA molecules to come together.
04. SDS: It is a detergent which destabilizes the phospholipid bilayer and also reduces the surface tension of water.
05. Sodium Acetate: Increases the salt concentration in the solution and brings about salting out of the DNA as it is insoluble at pH 5.3. It removes the negative charges on DNA so bases are in phenol form.
06. Ampicillin: It acts as a competitive inhibitor of the enzyme transpeptidase, which is needed by the bacteria to make their cell walls.It inhibits the third and final stage of bacterial cell wall synthesis in binary fission, leading to cell lysis.
07.Ethanol: a) Absolute ethanol: Removes the water of hydration. So the DNA cannot dissolve and sets free in the solution.
b)70% ethanol: Removes the impurities and traces of absolute ethanol.
PROCEDURE:
01. Appropriate calculations were done for respective salts.
02. The required amounts of the salts were measured.
03. Solutions were prepared according to their respective concentrations.
04. pH was set for the desired solutions and then the volume was made up with the help of distilled water.
05.For EDTA solution preparation, the salt after being weighed was dissolved in about 10ml of warm water. NaOH pellets were added to the solution, while keeping on magnetic stirrer, till the solution gets clear. The pH was set to 8.0.
06. The chemicals were all labeled properly and stored for usage.
Comment feed for this post
