20.109(S11): TA notes for module 2

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(Day 3)
Line 118: Line 118:
*Part 3
*Part 3
** 45 mL LB per team (need 42)
** 45 mL LB per team (need 42)
 +
** 25 mL pipettes
 +
** 50 mL tubes
 +
** 15 mL tubes
** 150 μL aliquots of AB25
** 150 μL aliquots of AB25
** a few Amp aliquots to share, 250 μL each
** a few Amp aliquots to share, 250 μL each

Revision as of 13:03, 15 March 2011

20.109(S11): Laboratory Fundamentals of Biological Engineering

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Contents

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
    • 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)
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