SEED/2009/Day 6

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

Instructor Post-prep

 * Store plates in fridge