Question

Why is the cloning reaction, transformation reaction, and pouring plates important for sequencing a plasmid? This is a lab-related question. We did cloning, transformation, and poured plates in lab, but why is this important for sequencing a plasmid? Please help me!!!

Answer 1

Plasmid

a plasmid is a small DNA molecule within a cell that is physically seperated from a chromosomal DNA and can replicate independantly. They are most commonly found as small, circular double stranded DNA molecules in bacteria.

Artificial plasmids are oftenly used as vectors in molecular cloning.

DNA sequencing provides the most complete characterization of recombinant plasmid DNAs. Using primers targeting the plasmid backbone and/or the insert sequence, the identity and order of nucleotide bases for any given DNA can be determined. In the process of cloning, sequencing allows users to confirm the DNA sequence of the insert, insert orientation, and to examine the junctions between the plasmids and insert the DNA. The size of DNA insert will dictate how many and which primers are necessary to determine the complete sequence. In cases where the complete insert sequence must be determined, it is advised to use primers targeting both the plasmid DNA ~ 100 - 150 bases outside the insert side and to insert DNA.

SIGNIFICANCE

In any cloning experiment the use of positive and negative controls is important. Without appropriate positive and Negative controls for your cloning and tranformation reactions it is very difficult to evaluate rhe result of clocloning event. These controls are indicators of enzyme activity in DNA preparation and transformation efficiency of competent cells. To ensure the efficiency of cloning reactions, each of the procedure are important.