Day 1: Plasmid transformation
In molecular biology, vector is a artificial medium which carries exotic genetic material from one cell to another in order to later express and replicate. There are four major kinds of vectors: plasmids, viral vectors, cosmids, and artificial chromosomes; plasmids are the most common kind. Some characteristics define a vector, such as an origin of replication (where the vector can replicate themselves autonomously inside its host, possessing a compatible site for genetic material, having the ability to clone in great amount, and retain genetic markers which help to distinguish recombinant cells from non-recombinant cells.
Plasmids, the vector chosen in this experiment, are generally circular, dual-standard DNA molecules and are independent from chromosomal DNA of any given cell because they have own origin of replication. Plasmids can not survive out of its host and it can not only replicate independently, but also add genetic information to its host which known as transformation.
When cells are placed in a 0 degrees Celsius CaCl2 solution, E. coli bacteria would become more permeable to the plasmid DNA. Then, after the cells are placed on ice along with the DNA. Then, after the cells are placed on ice along with the DNA, the solution would get heat-shocked at 42 degrees Celsius for about 2 minutes. This would promote the absorbance of the genetic material. The plasmid would then give the bacteria resistance towards a specific type of antibiotics, which could be used in the screening process.
The aim of this experiment is to transfer plasmids（hFXRα,FXRE,SV40,RXRα） to competent cells in order to greatly amplify plasmids.
Day 2: Plasmid amplification
Day 3: plasmid extraction
The aim of this experiment is to amplify the plasmid DNA via the shaking incubator, extract the plasmid DNA from the other proteins that might be present in the solution, and purify the solution in order to isolate the plasmid DNA.
Day 4: Cell recovery and culture
Culture HEK293 cells for 24 hours, prepare for cells transfection.
Day 5: Amplification, Extraction and purification of plasmid
In this experiment, the method of alkali cracking was used to prepare plasmids in small quantity. In the narrow range of pH between 12.0 and 12.5, the linear DNA double helix structure was unraveled and denatured, while the hydrogen bond of covalent closed-loop plasmid DNA would break, but the two complementary chains were coiled around each other and still tightly bound together. When ph4.8 acid potassium acetate was added to reduce the pH value of the solution, the plasmid DNA was rapidly recovered, while the chromosome DNA was large and difficult to be recovered. By centrifugation, most cell fragments, chromosome DNA, RNA and protein will be precipitated (under the action of SDS) to remove, while plasmid DNA remains in the supernatant. The purified plasmid DNA can be obtained by cation column adsorption and enrichment, then precipitation with isopropanol and washing with ethanol.
A large number of receptor cells and plasmids were obtained and successfully extracted
Day 6: Identification of plasmids by agarose gel electrophoresis
Different DNA, different molecular weight and different configuration, the mobility of electrophoresis is different (the migration rate is inversely proportional to the logarithm of molecular weight), so as to distinguish different zones, namely molecular sieve effect. Other factors affecting mobility include gel concentration, voltage and pH.
Nucleic acid banding observation is to use the combination of nucleic acid and nucleic acid dye, and the color development under ultraviolet light, with the aid of ultraviolet observation instrument.
It is proved that the molecular weight of the target DNA is equivalent to the theoretical value（hFXRα,FXRE,SV40,RXRα）.
Day 7: Protein expression and purification
Day 8: AlphaScreen-Based Protein-Peptide Interaction Assay
Day 9: Cell transient transfection and reporter gene experiment
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