BS5002 - Cellular Biochemistry (TERM 2 | WEEK 1 - Cloning a disease gene)
Key learning objectives list: LO1 - How to prepare 'donor DNA' for cloning LO2 - Understand the concept of gene libraries LO3 - Understand phage vectors used to create gene libraries LO1 - How to prepare 'donor DNA' for cloning Step 1: Choose a tissue The final product of transcription of DNA is mature mRNA. This type of mRNA has been fully spliced (introns excised and exons ligated), leaving only the coding sequence of the gene. Mature (spliced) mRNA. This mRNA can be translated directly to synthesise proteins in the ribosome. It is important to choose a tissue that expresses a fairly large level of mRNA from the gene of interest. Step 2: RNA extraction Whilst various methods exist, one of the most common methods for extracting total RNA from a biological sample is AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction). Step 3: mRNA purification Again, there are numerous methods for mRNA isolation from