User:Daisy.v.leon/sandbox

 DNA 

Transformation is when a recipient cell takes up and replicates exogenous DNA. Plasmids are used as cloning vectors and they are engineered to be 2.5-10 kbp, in order for the insert DNA size to be maximal. The time needed for replication depends on size of plasmid. The plasmids have one origin of replication. They can express drug resistant markers that replicated within their host cells.

Plasmid pBR322 is a cloning vector that is used to demonstrate an example of a Bacterial transformation experiment. BamHI cleaves pBR322, and generates inserts from foreign DNA. This is followed by annealing and ligation of inserts which creates the chimeric plasmid. This new plasmid transforms Ca2 -treated heat-shocked E. coli cells. This bacterial sample then is plated on a petri dish to be incubated at 37°C. When the incubation period has elapsed colonies of ampicillin resistant bacteria are present. Plating the bacterial sample on plates of tetracycline-containing media we can see colonies that are tetracycline sensitive. These colonies have tetracycline resistance and can be concludes that these colonies have plasmids with transformed foreign DNA inserts, because the control group that was not transformed, was not tetracycline resistant and could not survive in a tetracycline-containing media.

 Polymerase Chain Reaction 

Polymerase chain reaction is a method used to amplify the quantity of a desired DNA segment. The template for the DNA polymerase is the denatured DNA containing the specific desired segment. While two specific oligonucleotides are the primers for DNA synthesis, that are added 1000 times in excess. They catalyze the synthesis of the two complementary strands, which doubles its concentration in the solution. The DNA is then heated in order to separate the double strands and later cooled down to allow primers to bind both new and old strands. This is done through thermal cyclers that heat the reaction mixture in order to dissociate, cool, anneal, and perform as many rounds of DNA synthesis as required. Every round of synthesis theoretically doubles the amount of DNA present. In some studies it is noted that the theoretical amount of DNA present after 25 rounds should increase in concentration by 33 million but when measure was actually close to 1 million. Non-the-less 1 million is ample for gene isolation. This method effectively increases a small quantity of genomic DNA in a mater of hours.

 Alkaline Phosphatase 

An example of a reaction catalyzed by Alkaline phosphatase is as followed; CH₃OOOPO₃²- + H₂O CH₃OH + HPO₄²-, where Alkaline phosphatase has an uncatalyzed rate (Vu) of 1x10-¹⁵ sec-¹, catalyzed rate (Ve) of 14 sec-¹, and Ve/Vu of 1.4x10¹⁶.