20.109(S11): TA notes for module 2
From Course Wiki
Revision as of 16:21, 23 June 2014 by AgiStachowiak (Talk)
Contents
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 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 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.
- Prepare some aliquots of the miniprep reagents.
- 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)