User:Daniel A. Vargas/Notebook/General lab notebook/2015/08/07

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LCR results Restriction Enzyme Digest results Reagent Volume Expected: 1. MV10/gal4/atf2 1 = 7230 2. MV10/gal4/atf2 2 = 7230 3. MV10 = 5191 27 μL/lane; 1% agarose; Ladder DNA(plasmid) 3.0 μL 10X FD buffer 3.0 BsmBI 1.0 dH2O 20   27 μL --> 37°C/ 15 min. Nanodrop results Plasmid Conc. (ng/µL) St Dev (ng/µL) 260/280 MV10/gal4/atf2 1 10.03 3.20 1.99 MV10/gal4/atf2 1 12.02 3.63 1.98 MV10/gal4/atf2 2 147.770 5.04 1.908 MV10/gal4/atf2 2 146.57 4.13 1.907 Conclusion The first lane of the gel shows no plasmid and had a 10.03ng/µL concentration which would indicate there was actually no plasmid either isolated or present in the selected colony. However in lanes two and three bands are visible which means that there is plasmid present. The expected size of the band in lane two is 7,230 which was ran along side the bare backbone, not containing the insert, and both appear to be the same size. This fails to support that the ATF2 and Gal4DB/mCherry inserts were incorporated into the vector. The error made was in selecting only two colonies from the plate, selection of up to at least ten seperate colonies will increase the chances of finding a colony that contains the desired plasmid.