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:

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

Day 3

Materials required:

• Two days before lab, streak out the following strains:
  • AB25 = pIPTG-lacZ on Amp plate
• One day before lab, prepare O/N cultures of same
  • AB25 is for transfer function
• One day before lab, prepare O/N digests of pED-IPTG-INS backbone
  • 5 U each of XmaI/BamHI enzymes, 400-500 ng bkb per team

• Part 1
  • three half-gels per day, 0.7%
  • 25 μL digest aliquot per student
  • 10 μL aliquots of loading dye (Parts 1 and 2 need 5 μL total)
  • post gel plan at gel bench
  • clean spatula(s) out at transilluminator box
• Part 2
  • enzymes out on cold box partway through lab
  • 2-3 aliquots of NEB4 and BSA out on ice
  • undigested bkb aliquots out - two aliquots of 10 μL each, to share
• 15 mL conical of RNase free water out to use for Parts 2/4/5/6
• Part 3
  • 45 mL LB per team (need 42)
  • 25 mL pipettes
  • 50 mL tubes
  • 15 mL tubes
  • 150 μL aliquots of AB25
  • a few Amp aliquots to share, 250 μL each
  • 14 mm rb tubes out
  • a few tubes of 1 M IPTG to share
• Part 4
  • oligos and spec sheets out at their bench
  • a few aliquots of ligase buffer to share (10 μL needed per team)
  • heat block out, we start when everyone ready
• Part 5
  • 50 °C bath up front and ready
  • QIAEX II materials out and up front
  • help students before drying step as needed
• Part 6
  • Turn on spec
  • Spec UV lamp on toward end of lab
  • UV cuvettes out

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)