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V Fischeri assignment



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    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 else the cell and isolate the nucleic acid. After isolating the nucleic acid use an Rinse in order to isolate the genomic DNA of the Brio Fischer 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 Brio Fischer DNA as well as the page vector with the enzyme.

    Then use TO DNA aliases to legate the pieces of DNA to the vector. Then transform the legation 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 Brio Fischer legated to plasmid. Glowing colonies have the lug gene legated to the vector. Locate and streak a single glowing colony. Use PC to clone both the glowing colony and the Brio Fischer DNA. Sequence the product and finally 2) Legation: a) There will be 4 legations mixture reaction samples.

    Each of the All to LA tubes ad digested Brio Fischer DNA (or the insert), digested page (the vector), xx legation buffer. To eliminate volume as a factor the rest of the sample was filled to 30 LU with water. The main difference between each sample was that the insert: vector ratio. So each tube of -4) had different amount of inserts. This variation of the amount of insert Brio Fischer DNA was to find the optimum ratio in order for the insert to correctly legate to page vector. The 5th L tube had only digested page without any insert. B) c) 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 TTL O, TTL end, TO 0 T 2 end, until TO end. The main difference between the two is that the TO samples although digested does not contain any aliases so there should be linear page (seen at around 3200 BP) and linear Brio Fischer DNA. The T end samples contain the TO DNA aliases thus there will be a lot less if any linear page instead there will be a lot of supercilious legated page seen at around 2000 BP. There should also be some larger DNA bands that contain both the Brio Fischer insert

    DNA with the page plasmid. Using this we can see if the legation 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 legation mixture as in one the aliases was present where in the other one there was none present. The easiest way is to compare the linear and supercilious band as mentioned earlier. In TO samples there should be only linear page where in the mixture with aliases there will be supercilious page present. 3) Transformation – a) We had both a positive and negative cotton

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