Week 6

August 15-

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

August 16-

Digestion; Gel Electrophoresis and Purification

Ligation (10 minutes at room temperature)

Transformation (50 µL DH5α, 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 DH5α, 2 µL DNA, 37 degrees Celsius overnight, all carb)

August 17-

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 DH5α, 2 µL DNA, 37 degrees Celsius overnight, all carb)

Incubation in 3 mL of LB chlora overnight at 37 degrees Celsius and 220 rpm

August 18-

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