20.109(S11): TA notes for module 2

General notes

Key preparation:

Scheme:

Day-by-day

Day 1

Materials required:

• Two days before lab, streak out the following strains:
  • ABX8 = pEDL3/pCph8/pPLPCB, on Amp/Cam/Kan plate
  • ABX(22?) = pED-IPTG-INS, on Amp plate
• One day before lab, prepare O/N cultures of same
  • AB8 is for edge detection plates
  • AB22? is for first liquid culture experiment

Below are at set up at teaching bench unless otherwise noted:

• Equipment
  • Both water baths

• Cells
  • Aliquots of AB8 and AB?22?, labeled with strain and/or plasmid name, 1 per group

• Consumables
  • A few items should be at their benches, or the front gets too crowded; doesn't matter too much which
  • Bags of 14 mL rb tubes (56 tubes needed per day)
  • Pack of 50 mL conical tubes
  • 2 boxes of cuvettes
  • 5 and 10 mL pipets, pipet-aid
  • Empty Petri dishes (AT THEIR BENCH)
  • 15 mL conical tubes (AT THEIR BENCH)
  • Photomasks

• Reagents (see google doc for amounts not listed)
  • LB aliquots: ~30 mL per group (25 + for OD measurement + excess)
  • On ice, antibiotic and additive aliquots, about 1 per 2 groups
    • Plain ampicillin
    • AHL
    • IPTG
    • Amp/Cam/Kan cocktail

Day of Lab (T/W):

• Prepare supplemented LB medium (30' autoclave, 30' or 60' for pressure to go down in large or small autoclave, respectively) and cool in a 42 °C water bath for at least 1 hour
  • Can autoclave in one bottle, but need to simultaneously autoclave 1-2 more empty bottles to split media into (so a spill doesn't mean all is lost)
  • Prepare enough so have 1 plate per group, plus 1 for TA, plus 2-3 extra
• Turn on water bath so it has plenty of warm-up time, e.g. when begin autoclaving
• Turn spec. on

After Lab

• Turn water bath back off.

Day after Lab (W/R):

• Move plates and liquid cultures to 4 °C

How it went:

• Timing was fine, not overwhelming.
• Some folks mixed up the cells and/or antibiotics at first: set up one labeled station for liquid culture, and one for solid culture in the future.

Day 2

Materials required:

Day of Lab (R/F):

After Lab

Special materials

Cell strains

• NB399 = JW3367c (EnvZ-, lacZ-, Kan^R removed)
• NB435/AB? = pEDL3, pCph8, pPL-PCB (full ED system)
• NB? = light to lacZ only
• AB? = pED-IPTG-110
• AB? = pED-IPTG-112
• AB? = pJT104 (full ED system less AHL)