20.109(S11): TA notes for module 2

General notes
Key preparation:

Scheme:

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 &deg;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 &deg;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 &beta;-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 &mu;L digest aliquot per student
 * 10 &mu;L aliquots of loading dye (Parts 1 and 2 need 5 &mu;L 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 (enough for multi-rxn cocktail!) and BSA out on ice
 * undigested bkb aliquots out - two aliquots of 10 &mu;L 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 &mu;L aliquots of AB25
 * a few Amp aliquots to share, 250 &mu;L 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 &mu;L needed per team)
 * heat block out, we start when everyone ready
 * Part 5
 * 50 &deg;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 &deg;C

Day 4
Materials required:


 * Part 1
 * 4-5 aliquots of ligation buffer (10ish &mu;L), 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
 * Part 3
 * 4 LB-Amp plates/team
 * 5 ng/&mu;L plasmid, few 5 &mu;L aliquots
 * XL1-Blue tube/team (check stocks!)
 * 2.5 mL LB/team (can be in 4-5 aliquots)
 * burners filled with denatured ethanol
 * 42 &deg;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.

Day of Lab (T/W):
 * 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/&mu;L
 * Part 4
 * Plates at their benches.
 * Part 5
 * Sequencing tubes, caps, and water/primer mix.


 * Quiz ready
 * Turn on heat block at 42 &deg;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:


 * &beta;-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

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)