Results and Testing Chloramphenicol Plates
- Cells grew in both LB-only and LB+Chlor
- Possibly due to insolubility of antibiotic.
- Made LB Broth only (10 mL) and LB Broth + Chloramphenicol (10 mL Broth + 10 µL Chloramphenicol)
- Innoculated with DB3.1 cells
- Left to grow in Lagally Lab shaker till next morning.
Obtained results on Tetracycline plate
- retrieved plates at 13:06 from the incubator from Lagally Lab BioHazard room
- observed no growth on LE-tet plate
- observed cell growth on LE-only plate
- Conclusion: Tetracycline is effective
Transformation of Biobricks
- The GFP, pBAD, Terminator and J23100/RBS showed slow growth colonies, only becoming apparent this morning.
- The Tet Construction Plasmid and Kan Construction Plasmid showed no growth, will perform another transformation of these Biobricks
Transformation of Tet and Kan Construction Plasmids
- Transformation performed in DB3.1 cells
- Tetracycline and Kanamycin construction plasmids - 1 µL of DNA into competent cells stock
- On ice for 30 minutes
- Heat shock at 42.5ºC for 45 seconds
- 2 minutes on ice
- Added 400 µL of sterile LB broth to the 100 µL of glycerol stocks (in the same tube as the glycerol stocks). Total volume in microfuge tube should be 500 µL.
- Recovery for 2 hours at 37ºC
- Spread plated 100 µL of Tet-Construction Plasmid (Tet-Con) cells onto Tetracycline plates, and did the same for Kan-Con plates.
- Dried for 20 minutes.
- Incubated at 37ºC ON in AMBL.
Miniprepping of Amp Construction Plasmid
- Used 5mL of a 10mL overnight culture with the Invitrogen ChargeSwitch Miniprep Kit
- Followed protocol exactly, eluted in 100uL elution buffer.
- Final DNA yield was 6ug (61.2ng/uL as measured by Nanodrop)
- Somewhat lower than the typical yield of 25ug - not certain as to the reason