A new project that my supervisor Jim and I have been working on is to take a plasmid called pcDNA 3.1 with the BK gene and digest with restriction enzymes. By doing a restriction digest, we are trying to cut the BK at different sites known as Bam and Sma. By cutting at these different sites we are trying to make a fragment that is complementary to another plasmid known as pEntr. Once a fragment is in pEntr, through a very simple process known as a cloning reaction, we are able to take the gene in the plasmid pEntr and put it into various different plasmids such as dest 40, adenovirus, baculo virus, and many other different types of plasmids.
This is an easy way to take one gene fragment and use it in many different types of ways that is relatively simple and has a high transformation rate. Because I’ve been doing these pretty much all summer, I have become a “pro” at these and can practically do them without even looking at the instructions. During the cloning reaction, the destination vector, the gene, pEntr, and the LR clonease enzymes are mixed together in order to promote the Clonase reaction.
After a couple of incubations, the reaction mixture is put into E coli cells and after I heat shock and then let the cells rest before placing media that will allow the bacteria to feed and grow, the reaction mixture is plated on selective media in which antibiotics are used to sort between destination vector that were successfully transformed and those that were not.