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Team:Brown/Notebook/June22
From 2010.igem.org
Tuesday, June 22 2010
CaCl2 competence protocol
Zach created overnight cultures of E. coli on 6/21. We received 500 mL of XL1-Blue, 500 mL of BL-21 strains.
XL1-Blue = better for transformations
BL-21 = better at protein expression
- 9:30 AM – overnight culture was diluted 1:250 in LB; OD readings taken after 2 hours (every 20 minutes at 600 nm)
OD READOUTS
| Time | XL1-Blues | BL-21 |
|---|---|---|
| 11:30 | 0.058 | 0.120 |
| 11:53 | 0.081 | 0.201 |
| 12:13 | 0.122 | 0.314 |
| 12:35 | 0.122 | 0.431 spun @ 3000 rpm, 10min 4°C |
| 12:51 | 0.255 | |
| 13:13 | 0.373 spun @ 3000 rpm, 10min 4°C |
- Resuspended cell pellets in TFBI
- Protocol called for 100 mL TfBI per cell type
- Added 50 mL to BL 21
- This was half of the correct buffer amount
- Added 150 mL to XL1-B
- This was 1.5x the correct buffer amount
- Incubated on ice for 25 minutes.
- Spun at 3000 rpm, 10 minutes
- Respun XL1B at 5000 RPM, 5 minutes because it wasn’t fully pelleted.
- Resuspended each cell type in 10 mL TfBII
- Aliquoted into 1.5 mL microfuge tubes; placed in -80°C freezer (trays labeled with paper flags)
