G.tigrina Hox gene DthoxC insertion into prokaryote E.coli / (by UNIamCloning)/2011/10/05

DNA isolation/purification of G.tigrina

• Today we continued the process of extracting planarian DNA from our 0.025g sample that we collected yesterday (10/04/2011) and placed in the incubator. We manipulated the pH of the sample several times using a succession of buffers suggested by Qiagen DNeasy Blood & Tissue Kit, each time allowing "junk" to be flushed through a column membrane while the DNA stuck to the filter. This was done by centrifugation in a 1.5mL column. During the final spin, the DNA's pH was manipulated once more, this time allowing it to pass through the filter and collect in a labeled 1.5mL tube, extraction [GT.4]. The DNA extraction was checked using Qubit which said the sample's DNA concentration was too low to read. This may have occurred because the DNA concentration was so high it overwhelmed the mini column. For tomorrow, we will run another DNA isolation using two samples of 0.012g planarians (half the amount used in Gt.4). In one of the tubes, the amount of Buffer AE (the last buffer added during the DNA extraction) will be increased. Also, we will run another PCR reaction using an increased concentration of DNA (3µl instead of 1µl) and a decreased concentration of water for 2 samples of Gt.3 and 3 samples of Gt.4.