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. lsolate and streak a single glowing colony.
use PCR to clone both the glowing colony and the Vibrio fischeri DNA. Sequence the product and finally 2) Ligation: a) There will be 4 ligations mixture reaction samples.Each of the Ll to L4 tubes had digested Vibrio fischeri DNA (or the insert), digested pGEM (the vector), 5K 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(l -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 insert. b) ) The main different between the lanes is TO and Tend samples.There is a TO and Tend sample that is paired up as organized into Tl O, T lend, T2 0 T 2 end, until T5 end. The main difference between the two is that the TO samples although digested does not contain any ligase so there should be linear pGem (seen at around 3200 bp) and linear Vibrio fischeri DNA The T end samples contain the T4 DNA ligase thus there will be a lot less if any linear pGEM instead there will be a lot of supercoiled ligated pGem seen at around 2000 bp.
There should also be some larger DNA bands that contain both the Vibrio ischeri insert DNA with the pGEM plasmid.IJsing this we can see if the ligation worked. The easiest way of doing this is compare the To and T end bands. They should be different on the gel despite coming from the same original ligation mixture as in one the ligase was present where in the other one there was none present. The easiest way is to compare the linear and supercoiled band as mentioned earlier. In TO samples there should be only linear pGEM where in the mixture with ligase there will be supercoiled pGEM present.
