Miniprep of Amp Construction Plasmid and Chlor Construction Plasmid
- Centrifuged 5mL of each construction plasmids in a 1.5mL centrifuge tube for 4 runs at 14000rpm (Galaxy 16) for 10 min.
- Add 250uL of Resuspension Buffer with RNAase A and resuspend by vortexing
- Add 250uL of Lysis Buffer and invert 10 times
- Inncubate at room temperature for 5 min
- Add 250uL of Precipitation Buffer and invert 10times
- Transfer solutions to 2 filter mini columns some precipitate was not transfered because because it was sticking to the tube and pipet
- Wait 2 minutes
- Centrifuge at 4100rpm (1350xg) for 1 min
- Remove Clarification column
- Add Wash Buffer 1 (750uL) to column
Centrifuge at 10200rpm (8500x g) for 1 min
Add 250uL of Wash Buffer 2 and centrifuge at 10200rpm
- Insert column nto 1.7 elution tube and add 100uL Elution Buffer
- Spin at 10200rpm for 1 min. Store at 4°C
Results of Miniprep
| Amp Construction Plasmid
| Chlor Construction Plasmid
NOTE: All results were found using a nanodrop with 1uL of sample
Making Competent BW27783 and DH5α cells
- Inoculate 5mL overnight culture and grow at 30°C.
- Dilute 0.5mL overnight culture into 50mL LB and incubate at 30°C, shaking vigorously.
- Harvest BW27783 at Abs600nm = 0.482 and DH5α at Abs600 = 0.390.
- Centrifuge at 1600g for 7 minutes (4°C) - (Using AMBL centrifuge, 4500rpm).
- Wash gently in ~20mL COLD filter sterilized 0.1M CaCl2.
- Spin down gently: 1100g, 5 minutes, 4°C - (Using AMBL centrifuge, 3700rmp).
- Resuspend in 12.5mL cold 0.1M CaCl2.
- Keep on ice 40 minutes.
- Spin down: 1100g, 5 minutes, 4°C.
- Resuspend in 2-2.5mL cold 0.1M CaCl2.
- Store over night at 4°
- Use 100uL aliquots, store at -80°C?.
Resuspension and Transformation of BioBricks
- Worked with Karen and Amelia
- Resuspended BioBricks into 15 µL of sdH2O in new PCR tube
| J23100 promoter