Of protein expression - Lab Report Example

Download file to see previous pages Therefore, in some cases, the given GFP gene can get attached to the given end of a gene required to encode a given protein of interest. Cloning Cloning by definition involve making an identical copy of the original. Cloning is usually considered as an amplification process, since it entails making of identical copies from a single copy. Therefore, cloning DNA simply involves recombinant DNA. Often, there exits two different DNAs which are required in the process of cloning. One of them include an insert (the given molecule to be cloned), then secondly, a vector (cloning vehicle). In addition, a white/blue selection vector usually remains as the common plasmid cloning vector type. The vector contains an original of replication, allowing plasmid replication to occur, hence providing the actual amplification step that allows selection of a given bacteria which contain the vector. Furthermore, it contains the E.coli lacZ gene that allows for selection and insertion of DNA fragment into a given vector. Therefore, the lacZ gene contains an MCS (multiple cloning site), an oligonucleotide sequence that exists in series with other restriction endonuclease recognition sites which are usually arranged in tandem providing the same reading frame like the lacZ gene. Therefore, this is surely the heart around white/blue selection. Usually, the lacZ gene often codes for (B-gal) B-galactosidase, a given enzyme which cleaves lactose galactoside bond. Furthermore, it also can cleave galactoside in X-gal, an artificial subtract. X-gal, if added in a media containing growing bacteria, usually turns blue when cleaved. When a DNA fragment gets inserted into the MCS, usually the lacZ gene becomes disrupted, resulting to its inactivation, leading to B-gal not able to cleave X-gal, causing presence of white colonies. Vector and the insert often get cut by the same destruction enzyme leading to production of single-stranded sticky ends. In this case, the variable entails the ability for it to select various enzymes which recognize difference restriction sites. After this, ligation is performed to generate fewer multimers. Next, entail the transformation process of which involve DNA introduction into a given host cell. In E.coli, DNA has to be forced in it since it does not undergo a normal physical transformation. Usually, two methods can be used for transformation: on entails washing the given bacteria with very high salt concentration of calcium chloride, or by passing current through it (electroporation). E.coli contains lacZ gene hence function with the white/blue system. The commonly used strain is DH5 alpha. Usually, transformation produces multiple bacteria types, some of which are unwanted since they contain vectors without inserts. Then, the transformed bacteria are usually placed in an agar plate which contains ampicillin and IPGT (Isopropyl Thiogalactoside), a B-gal inducer. Ampicillin hence kills bacteria which would not have transformed. The remaining population usually thrive to produce white colonies and blue colonies. It is necessary to remove false positive clones. Materials and Methods: Materials: The following materials were required for the experiment to be successful. 2µl HindIII/ EcoRI cut plus cleaned PUC19 vector 5 ...Download file to see next pagesRead More