20.109(S11): TA notes for module 2
From Course Wiki
Revision as of 14:06, 10 March 2011 by AgiStachowiak (Talk)
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:
Most components for β-gal assay will be one aliquot per team, for them to pick up at the front bench (exceptions noted below).
- Z-buffer, this year a 15 mL conical full per group
- 0.1% SDS, 0.25 mL/team
- Na2CO3, 3 mL/team
- ONPG, ~1.1 mL/team to be safe, *or* just have extras ready
- aliquots are 1 mL, and some students may run out
- stock is 4 mg/mL in water
- Chloroform should be in an easily accessible container (that they don't have to tip) in the hood.
- 5 mL total needed, ideally in 2 stations with 2 eppendorfs each (two groups can fit in the hood at once).
Day of Lab (R/F):
- Quiz ready
- Thaw ONPG on ice
- Keep an eye on students so they complete their designs by around 2:30/2:45 pm.
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)