Week+September+12

__September 14, 2011__ Today was our first day at Jordan Hall. We discussed an overview of the lab materials with which we will be working. In the lab, we will be using a portion of jellyfish DNA, the Green Flourescent Protein (GFP) possessing gene to make the bacteria that will carry the gene. This lab we will be doing has 4 parts. 1. PCR (Polymerase Chain Reaction) This process will isolate the pure GFP In PCR dNTP's (free-floating nucleotides) are u‍‍‍‍sed to bond with the primers ‍‍‍‍ At the end of PCR, you have pure GFP DNA 2. Ligation The GFP DNA is added into a cut vector to form a plasmid. The vector is cut using rescriction enzymes. Restriction enzymes can be of two forms. 3. Transformation Bacteria cells must be stressed in order to insert the plasmid into the bacteria. To stress the cells, two forms of shock can be used. In this lab, heat shock is used. By heating the bacteria to the right temperature, they become porus a nd begin to pull in the surrounding, The plasmids are inserted because they are pulled in by the bacteria. After the heating and insertion, the bacteria must be cooled ‍‍‍‍so that replication can start at the origin of replication on the plasmid, ‍‍‍‍ 4. Plating and Selection The bacteria is plated, on a plate containing antibiotic (ampicillin in this lab) to grow. ‍‍‍‍Colonies of thousands of bacteria with the plasmid (which is ampicillin resistant) will grow on ampicillin plates. All other bacteria will die, leaving only the desired bacteria.‍‍ When the bacteria grows, it is possible to observe ‍‍‍‍if the GFP was properly made into bacteria containing the plasmid through the ampicillin selection, and if the bacteria was able to make GFP protein from the GFP DNA in the plasmid by seeing if the bacteria expresses the green color of the GFP.
 * Denaturation: breaks the Hydrogen bonds of the dsDNA (@95 degrees C)
 * Annealing (of Primers): Forward and Reverse primers join to DNA ‍‍‍at the site where the dNTPs wil make copies in elongation. ‍‍‍‍ (* to make primers for the DNA, the DAN sequence must be known) (@ 50-60 degrees C)
 * Elongation of DNA: Using Taq polymerase, the dNTPs will make copies of the DNA. When the process is done, you are l‍‍‍‍eft with pure GFP ‍‍‍‍ (@ 70 degrees C)
 * Blunt cutting enzymes
 * Sticky end cutting enzymes
 * Heat Shock
 * Electric Shock