Genomic Library Case Study

1) A genomic library is a collection of identical recombinant vectors each containing different insert of DNA where each vectors representing the entire genomic DNA of a single organism. In order to create the genomic library we first have to lyse the cell and isolate the nucleic acid. After isolating the nucleic acid use an RNase in order to isolate the genomic DNA of the Vibrio fischeri and then purify the DNA via phenol: chloroform purification. The next step is to use a RE in this case Sal I and treat both the Vibrio fischeri DNA as well as the pGEM vector with the enzyme. Then use T4 DNA ligase to ligate the pieces of DNA to the vector. Then transform the ligation mixture and the competent cells and create a population with various insert: vector ratio. Any cell that gives rise to a colony has taken up a plasmid and any white colonies has the Vibrio fischeri ligated to plasmid. Glowing colonies have the lux gene ligated to the vector.Isolate and streak a single glowing colony. Use PCR to clone both the glowing colony and the Vibrio fischeri DNA. Sequence the product and finally
Each of the L1 to L4 tubes had digested Vibrio fischeri DNA (or the insert), digested pGEM (the vector), 5x ligation buffer. To eliminate volume as a factor the rest of the sample was filled to 30 uL with water. The main difference between each sample was that the insert: vector ratio. So each tube of L(1-4) had different amount of inserts. This variation of the amount of insert Vibrio fischeri DNA was to find the optimum ratio in order for the insert to correctly ligate to pGEM vector.The 5th L tube had only digested pGEM without any