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Purdue/23 June 2008
From 2008.igem.org
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Looked up QC information for the SOS and LacZ parts (J22106 and I732005). SOS is OK and consistent, but I732005 is Inconsistent and BAD for all analyses.
Found new part that is more consistent and OK:
- I732017: LacZ (full-length) + RBS in front
- Location: Plate 1009, Well 11H
- Plasmid: pSB1A2 (AmpR)
- RBS efficiency = 1.0 (based on Elowitz 1999 Repressilator)
Edited by Janie Stine
Transformation of LacZ and SOS parts
- Used OneShot MAX Efficiency DH5alpha-T1 cells (Invitrogen) and used a combo of their protocol and the iGEM protocol
- Thawed on ice
- Cut out parts from filter paper, warmed in 5 uL of TE 20 min.
- LacZ/RBS (I732015), plate 1009, well 11H, plasmid pSB1A2
- SOS promoter (J22106), plate 1003, well 12F, plasmid pSB1A2
- Added all TE/DNA (~5uL) to 25uL of cells for each part
- Let sit on ice 30 min. in 2 mL Eppendorfs
- Put cells in 42C water bath EXACTLY 30s (no mixing or shaking)
- Place on ice
- Add 250uL warmed SOC to each vial
- Shake EXACTLY 1 hour at 300rpm and 37C
- Spread 200uL from ea. vial onto LB plates (store remainder at 4C)
- Invert plates and let incubate at 37C until we leave (to get a kick-start to their growth)
- Let incubate at room temperature overnight (We can't get to the cells for more than 14 hours, so we're forcing them to grow more slowly)
Edited by Janie Stine
