The first step in running a restriction digest on a plasmid is to select the restriction enzymes to digest the plasmid. To determine which restriction enzymes will cut the DNA sequence, I used SnapGene software. The second step is to determine an appropriate reaction buffer by reading the instructions for the enzyme. Because I conducted a double digest, I had to select a buffer that worked best for both enzymes by looking at the NEB Compatibility Chart, which led me to choose the CutSmart buffer, because both the ECOR1 and XBA1 enzymes could be digested at 65 degrees Celsius by the CutSmart buffer. The digestion reaction consists of the DNA, restriction enzymes, dye, nuclease free water, and the CutSmart buffer. This reaction is gently mixed by pipetting. Afterwards, the tubes were incubated at 37 degrees Celsius for 1 hour. Then, to visualize the results of the restriction digest, conduct gel electrophoresis. The results of the gel electrophoresis will be discussed on Tuesday when I return to lab. Until then, I will keep researching on Dact1 and its relevance to neural crest formation.
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Prachi MishraOh hi there! ArchivesCategories |