Team:UT Dallas/week6

PCR of ptetR, RBS, LuxI, term
Gel electrophoresis and purification
Glycerol stock and miniprep
Incubation in 3 mL LB can overnight at 37 degrees Celsius and 220 rpm
Test digestion for 1 hour and 30 minutes at 37 degrees Celsius
Gel Electrophoresis
Test Digestion Observation:
     Lane 5 gave positive results for FGF-R: 2 bands observed at
     predicted bands size. Lane 4, 7, 9 were all negative bad results because EcoRI was used but only 1 band showed.
     This suggests that FGFR was not inserted. Lanes 10, 11, 12 were CheZ* and all matched negative control.
     Results are bad for CheZ*- will be regrown/miniprep/ligated.
Incubation in 3 mL LB chlora overnight at 37 degrees Celsius and 220 rpm

8/16/11

Digestion; Gel Electrophoresis and Purification
Ligation (10 minutes at room temperature)
Transformation (50 µL DH5a, 2 µL DNA, 37 degrees Celsius overnight, all carb)
Re- incubating – taking 20 µL of yesterday’s incubations and placing them in 3 mL of LB and appropriate
Transformation (50 µL DH5a, 2 µL DNA, 37 degrees Celsius overnight, all carb)

8/17/11

Glycerol stock ToxR and CheZ* colonies (2nd attempt)
Miniprep
no results on gel
Incubate in 3 mL LB carb overnight at 37 degrees Celsius and 220 rpm
Transformation (50 µL DH5a, 2 µL DNA, 37 degrees Celsius overnight, all carb)
Incubation in 3 mL of LB chlora overnight at 37 degrees Celsius and 220 rpm

8/18/11

Glycerol Stock- 350 µL cells and 150 µL 50% glycerol
Miniprep
Test digestion
Digestion, gel electrophoresis and purification
Glycerol stock colonies 4 and 5 CheZ*
Miniprep
Test digestion and gel electrophoresis
Ligation at 16 degrees Celsius overnight
Incubation in 3 mL LB carb overnight at 37 degrees Celsius and 220 rpm
Plate (507011) from Agar Stab

Glycerol Stock + Miniprep: pTet+mRFP, RBS-TetR-Term
Incubation: ctx-GFP (LB Kan)