SEED/2009/Day 6
From OpenWetWare
Morning
- set up 5ul ligations with plasmid, each of 6 promoters, plus one RBS.reporter
- 0.5ul T4 ligase buffer
- 10ng plasmid
- 10ng RBS.reporter
- 0.2ul promoter
- 0.25ul T4 ligase
- Incubate at room temperatures over lunch
- pour plates (LB + amp + IPTG + X-gal)
Afternoon
- Do quiz on units
- Individual meetings with instructor to discuss final projects
- transformation of 6 ligations + 10pg pUC19 into chemically competent E. coli Top10 cells
- use 100ul of cells and 2ul of the ligation mix
- Leave on ice for 30 minutes
- Heat shock for 45 sec @ 42C
- Place back on ice
- Add 300ul SOC
- Incubate @ 37C for 30 min
- plate entire transformation mix
- Incubate at 37C
Instructor Preparation
- oligos with different promoters from this set registry:Part:BBa_J23101 pre-cut with EcoRI/SpeI
- T4 PNK top/bottom oligos
- 10ul: 1ul T4 ligase buffer, 1ul 50uM oligo, 8ul water, 0.25ul T4 PNK
- 4h@37, 10m@65
- mix and anneal pairs
- T4 PNK top/bottom oligos
Instructor Post-prep
- Store plates in fridge