The main objective of this experiment is to study the structure of a region of the RyR1 receptor to understand its molecular function in structural biology. Analysis of the RyR1 receptor will be performed by generating a construct pET19-RyR11-536 for insertion into E.coli bacteria. The bacterial colonies grown will then be tested for proper transformation and proper expression conditions so that further analysis can be performed. If possible, one of the important things to do before performing an experiment is to find a method to visualise the experiment in order to check for possible mistakes and see if it’s feasible. Especially in the case of cloning, the restriction enzyme needs to be tested so that the digestion of the product and the plasmid can proceed successfully. In this scenario, the software Serial Cloner was used to check the virtual PCR and ligation of the construct to see whether the restriction enzymes that were chosen, Ndel FD and BgIII FD for the PCR product and Ndel FD and BamHI FD for the plasmid, were suitable for ligation of pET19 plasmid and RyR11-536 PCR product. …show more content…
The first thing to do is to proceed with the mass cloning of RyR11-536 DNA by PCR to amplify the DNA for later use. The PCR product was then purified to remove unused dNTP, template and primer so that the spectrophotometer will not read the free nucleotides and provide an incorrect measurement. After obtaining the readings at the required wavelengths, the concentration of the PCR product was determined so that further calculations can be made to figure out the volume of product necessary for digestion with the restriction endonuclease. The ratio obtained by the readings also allow for an estimate on the sense of purity of the PCR