Difference between revisions of "20.109(S11): TA notes for module 2"
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* '''Two days before lab''', streak out the following strains: | * '''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 | ** ABX(22?) = pED-IPTG-INS, on Amp plate | ||
* '''One day before lab''', prepare O/N cultures of same | * '''One day before lab''', prepare O/N cultures of same | ||
Line 75: | Line 75: | ||
'''Materials required:''' | '''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):''' | '''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 CaCl<sub>2</sub>, 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 | ||
+ | * <font color=FF3300>Turn on heat block at 42 °C.</font color> | ||
+ | * <font color=FF3300>Agi fill out sequencing form as they get close and be sure one of us brings it in by 5 pm (5:30?).</font color> | ||
+ | |||
+ | '''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''' | '''After Lab''' |
Latest revision as of 21:05, 28 July 2015
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 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.
- 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)