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:
  • AB8 = 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.
• Announce that high activity samples should probably take 1-2 min, and low activity samples should be incubated for some reasonable time like 10 min.

After Lab

How it Went

• Add taking photo of plate to protocol

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 large aliquots of NEB4 (enough for multi-rxn cocktail!) 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 up front 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

After Lab

• Spec off
• Next day, cultures to 4 °C

Day 4

Materials required:

• Part 1
  • 4-5 aliquots of ligation buffer (10ish μL), water, plus their DNA on 4 shared ice buckets
  • box of cuvettes also 4 shared ones in usual configuration
  • will come up one at a time for T4 ligase to teaching bench (keep in freezer otherwise) with the fine pipet, also can check calcs with us
• Part 3
  • 4 LB-Amp plates/team
  • 5 ng/μL plasmid, few 5 μL aliquots
  • XL1-Blue tube/team (check stocks!)
  • 2.5 mL LB/team (can be in 4-5 aliquots)
  • burners filled with denatured ethanol
  • 42 °C heat block up front, on
• Part 2: all as one aliquot per team
  • UPDATE #s FOR 15 SAMPLES, NOT 9
  • 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

After Lab

• Next day move colonies to fridge.

Day 5

Materials required:

• One day before lab, pick 3 colonies for each team from their bkb+ins plate.
• One day before lab, prepare a few tubes of JW3367c cells (no antibiotic in the LB).
• Morning of lab, prepare one tube per team (plus 1-2 extra) of sub-cultured JW3367c cells.
  • About 2 hours of growth from 0.12 OD is good. Take into account time for pre-lab lecture! Starting right at noon probably okay if tubes already ready/labeled, LB measured.

Up at teaching bench unless otherwise noted.

• Part 1
  • LB aliquots for measuring OD
  • 5 mL aliquots of CaCl₂, one per team on 4 shared ice buckets at their benches
• Part 2
  • Prepare some aliquots of the miniprep reagents.
    • Will start at similar but not identical times, so 1 per 2 groups okay.
• Part 3
  • 4 LB-Amp plates per team
  • LB, serological pipets, and pipet-aids up front for them to prepare liquid culture tubes for us
  • Transformation spreading/sterilization materials up front.
  • On their shared ice buckets, aliquots of pED-IPTG-INS at X ng/μL
• Part 4
  • Plates at their benches.
• Part 5
  • Sequencing tubes, caps, and water/primer mix.

Day of Lab (T/W):

• Quiz ready
• Turn on heat block at 42 °C.
• Agi fill out sequencing form as they get close and be sure one of us brings it in by 5 pm (5:30?).

After Lab

• See above re: sequencing rxns.

Day 6

Materials required:

Day of Lab (R/F):

• Quiz ready

After Lab

Day 7

Materials required:

• β-gal assay materials - see google doc for updated volumes (assuming 17 samples per team, will mean some excess)
  • one aliquot per team for most components (CHCl3 in hood)
• illuminator, focusing image, and camera available on far bench (across from NK)
  • charge camera night before

Day of Lab (T/W):

• Quiz ready

After Lab

Day 8

Materials required:

• None, analysis day.

Day of Lab (R/F):

• Quiz ready (MAYBE NOT)

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)