G.tigrina Hox gene DthoxC insertion into prokaryote E.coli / (by UNIamCloning)/2011/11/29

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Concentration Check and Ligation of ATF1, GFP, and Vector

  • Today Alex and I ran a gel using FastRuler DNA ladder, low range (we used 10µl and 5µl of the ladder) to check the concentrations of ATF1, GFP, and the plasmid. The concentration of each appeared to be at about a 1:1:1 ratio and was estimated to be at a concentration (comparing it to the brightness of the ladder) of 50ng/µl. The vector is approximately 2000bp, ATF1 is about 1500bp, and GFP is about 750bp. Therefore, 3/4 the amount of vector DNA will be used for ATF1 and 1/2 the amount of ATF1 DNA will be used for GFP (this is based off the ratio of base pairs). We prepared four ligation reactions one of which was a negative control. The negative control was composed of 4µl vector, 4µl water, 1µl 10X ligation buffer, and 1µl ligase. Our optimal concentration ligation reaction was made of 4µl vector, 3µl ATF1, and 1µl GFP (also included 1 microliter each of buffer and ligase). The final two reactions had 1µl vector, 5µl ATF1, 2µl GFP, and 1µl each of buffer and ligase and 1µl of everything plus 5µl water. On Thursday, we will transform the four reactions.