Haynes Lab:Notebook/Engineering PC-TFs/2012/04/12

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Summary

  • Retrieve mini preps for flyPCD assembly from freezer and thaw at room temperature.
  • Also let restriction enzymes thaw.
  • Do restriction digest (EcoRI/PstI)Diagnostic digest
' ' 15 µl Total Master Mix
1 rxn x 9
DNA plasmid 3 --
enzyme 1 1 9
enzyme 2 1 9
10x buffer 1.5 13.5
dH2O 8.5 76.5
  • Aliquot 12 μL to each tube. Then add 3 μL DNA to each tube.
  • Place each tube in heat block 37°C.

Follow steps for gel electrophoresis.

  • Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
  • Fill gel flask with up to 60 ml of TA buffer.
  • Create 1% gel by putting .6 grams of agarose into flask.
  • Microwave agarose solution for 40 seconds
  • Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
  • When flask is taken out of microwave, make sure that the agarose is completed dissolved.
  • Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
  • Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
  • Pour gel into tray.
  • Wash the agarose gel flask.

Attempted to prepare gel in tray. Unforunately, this ended up being a struggle. I messed up setting up the wells twice and this caused me to not complete my diagnostic digest.