Overview
Wirte an introduction explaining the folloing concept with correct citations. In this experiment, mini-prep protocol was used to isolate the plasmid DNA from the bioluminescent clones. To confirm each colonies contains lux+, the isolated plasmid vector was digested with Sal I restriction enzyme and the digested DNA was analyzed using gel electrophoresis. Vector and insert DNA were digested. The restriction fragment luxAB was excised from the gel, purified, and inserted into a vector with ligation reactions. The subcloned DNA was then used to transform competent cells, and the clones was screened for target DNA. LuxA gene was amplified using different DNA sources as the template.Sequencing reactions using the plasmid DNA from luxAB colonies, lux- colonies, and lux+ colonies were performed to determine the sequence of each DNA sample.
Introduction for a Lab report, give background information on:
Plasmid DNA isolation
Retraction mapping
Subcloning
Colony hybridization
PCR (regular and fluorescent)
What is the purpose of each PCR component? Buffer - dNTP mix - Primers –DNA polymerase – DNA template –
What steps are programmed into a thermocycler for a PCR reaction? What is the purpose of each step? How is the annealing temperature determined? How is the length of time for primer extension determined?
Which lux operon genes encode proteins that catalyze the reaction responsible for bioluminescence? What is the function of the other lux operon genes?
Sequencing
How are sequencing reactions conducted? What are the necessary components for a sequencing reaction? Compare and contrast with PCR.