2 Outline the main requirements to successfully clone a small PCR generated DNA fragment into pGEX with a view to cloning a small section (218bp fragment) of the Insulin Gene and obtain a purified insulin (53 amino acid portion) protein product.
Answer to include:
• Using whole blood as a starting material description of Red cell lysis, white cell lysis, protein precipitation, nucleic acid precipitation. Answer to include the composition and function of each solution in the extraction process (7.5 marks)
• Quantification and analysis of extracted DNA. Assessment of DNA integrity by Agarose Gel Eelectrophoresis (7.5 marks)
• Overview of the Polymerase Chain Reaction (PCR) with specific reference to Bioinformatics of primer design (with EcoR1/Xho1 restriction enzyme (RE) sites), NCBI site usage, Primer3© primer programme usage and primer ordering from Sigma Aldrich (15 marks)
• Analysis of PCR Product. Restriction Enzyme digestion of plasmid and PCR Product, T4 DNA ligation of plasmid and PCR Product and bacterial transformation of Recombinant Molecule into BL21 E. Coli (to include experiment performed in the laboratory) (5 marks)
IPTG induction of GEX-INS, Sepharose affinity chromatography and Thrombin cleavage to obtain the purified 53amino acid protein of the insulin protein! (5 marks)