Today we ran our purified plasmids and PCR product that were cut with corresponding restriction enzymes through the Qubit machine to determine the concentrations of all samples for both their 1st and 2nd elutions of purification. Calculations were then done to determine molar ratios since the plasmids are ~2400 bp and our pcr product is about 1280 bp. This makes a volume ratio of 1:2 (Plasmid to gene) in order to prepare a 1:1 molar ratio. The ligation protocol was followed for all three plasmids with our gene in a 1:1 and 1:3 ratio for each. A negative control was also done for each. The samples were left on the counter to incubate and ligate together until next class when the transformation protocol using competent cells will be completed.
Reference article: Evolution of steroid-5-alpha-reductases in comparison of their function with 5ß-reductase DOI: 10.1016/j.ygcen.2009.08.004