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

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

LCR results

  • Restriction Enzyme Digest results
Reagent Volume Expected:
1. MV10/gal4/atf2 1 = 7230
2. MV10/gal4/atf2 2 = 7230
3. MV10 = 5191
Today's gel
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.