20.109(S14): TA notes for module 2: Difference between revisions
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'''Materials required:''' | '''Materials required:''' | ||
*Part 1, flow cytometry | |||
**several ice buckets | |||
**purple tube holders (from 15 mL conicals) pre-chilled in fridge or on ice | |||
**flow cytometry tubes with strainers, also pre-chilled | |||
**pre-warmed PBS, phenol-red-free trypsin/EDTA, OptiMEM | |||
***one aliquot per team | |||
**<font color=orange>well in advance: teaching faculty lipofections should be done same day as student ones</font color> | |||
***single color controls, etc. | |||
*Part 2, inhibitor dose response after irradiation | |||
**<font color=orange>in advance: plate one T25 per irradiation condition</font color> | |||
**Compound 401 prepared in ethanol | |||
'''Day of Lab (R/F):''' | '''Day of Lab (R/F):''' | ||
*Pre-part 1 | |||
**TA collects teaching faculty samples | |||
*Part 1 | |||
**TA brings ready samples to flow facility as needed | |||
*Part 2 | |||
**might be done differently in S15 | |||
**TA/instructor trypsinizes cells, Samson lab person irradiates cells | |||
**help students implement their plan smoothly | |||
'''After Lab:''' | '''After Lab:''' | ||
'''How it went:''' | '''How it went:''' | ||
Flow times were sufficient and students got out on time as well. For 6 groups, did 2:30-4:30; for 9 groups, did 2:30-5. Had plenty of time for analysis, etc. this way. | |||
===Day 7=== | ===Day 7=== |
Revision as of 08:08, 18 June 2014
General notes
See also Dropbox Excel document for aliquoting amounts.
Scheme: Each pair of students will test K1 and xrs6 cells for Ku80 production. They will then assess the ability of K1, xrs6, and DNA-PKcs inhibited cells (K1 treated with Compound 401) to repair DNA damage via plasmid based assay and flow cytometry.
Key preparation:
- Lost of cell culture in this module. At times, both K1 and xrs6 need to be grown.
- Prepare cut DNA – one of each type – in advance, in case student yields are low.
- In fact, I miscalculated amount of DNA needed and all students had to use TA stocks. Rethink for S15. (Potentially for the best due to reducing sources of error, but…)
- Large preps of uncut endotoxin-free DNA can readily be purchased from Genewiz.
- If MCS design will be changed for S15, the design should be tested in advance.
- Briefly, insert is ordered from Genewiz, along with amplification primers from IDT; amplify insert for best cloning yield; restriction digest, then sequence, to identify a good clone; order stocks from Genewiz.
Note to TA:
- While you read the wiki, test each link, and make any link descriptions bold that are not already.
- As you read the protocols, add reagent calculations to the Dropbox Excel doc as needed.
Day-by-day
Day 1
Materials required:
All for Part 3, cell plating for Western.
- Two T25s per group, one of K1 and one of xrs6.
- Flasks for next day seeded at 600,000 cells
- Flasks for day after next, seeded at 400,000 cells
- Flasks for 3 days in future, seeded at 200,000 cells
- One(?) shared aliquot per team
- PBS, CHO media, and 0.25% trypsin
- A few aliquots of Trypan Blue at the scopes
- Equipment to have out
- 6-well plates
- conicals
Day of Lab (T/W):
- No Quiz
- Partially prepare TC hoods (vacuum lines and some equipment).
- Warm up media, PBS, and trypsin half hour before students use reagents.
- During class, assist students with Part 4 – get familiar with the questions in advance.
After Lab:
How it went:
Day went pretty smoothly; not many questions on Part 4 exercise.
Day 2
Materials required:
- Part 1, cell lysate prep
- In advance: pre-chill scrapers in fridge
- Ice bucket at each bench, with
- two empty epps
- RIPA buffer (exactly 250 uL)
- protease inhibitors (5 uL) – thawed last minute
- PBS (~4 mL)
- Part 2, protein measurement and prep
- Cuvettes at front bench
- Precision Red reagent aliquots, ~ 3mL
- at front bench or their benches, either way
- in advance: to room temperature
- Water for diluting lysates
- Part 3, PAGE
- Ice bucket on front bench with Kaleidoscope ladder aliquots
- Inside fume hood
- in advance: Boil water with chips at about level 5
- Laemlli aliquots and box for venting B-merc tips
- At gel bench
- Tons of well-mixed TGS prepped in advance
- Gel chambers set up – make sure to remove strip from bottom!
- Also remove combs and rinse wells with running buffer
- If class size permits, have an extra gel chamber/gel set up for practice
- Part 4, transfer step
- In advance: freeze ice packs
- Cassettes, pads, filter paper
- Membranes, scissors at a cutting station
- Green gel openers
- Lots of transfer buffer
- Blocking buffer (S14 used Odyssey)
Day of Lab (R/F):
- Part 1: take student samples to cold room centrifuge as needed
- Part 2: store excess lysates at -80 °C until Western results are in
- Part 3:
- TA trains at hood -- cap covers, etc.; instructor trains at PAGE loading
- Check gel progress early on -- check for buffer issues (high enough, well mixed) or gel issues (strip removed) if running strangely
- Part 4:
- Pre-soak ScotchBrite pads in cold transfer buffer
- Also pre-soak filters if using the thinner DAL ones, rather than thicker Bio-Rad ones
- Explain transfer in advance to keep things moving
After Lab:
Move their blots to blocking buffer.
How it went:
W/F students out b/w 4:25 and 5:15 (most at 5 pm). Blocking transfer completed by 6:45 pm.
Day 3
Materials required: None, your brains :)
Day of Lab (T/W): First quiz
After Lab:
How it went:
Day 4
Materials required:
- Part 1, digests
- Aliquot exactly 3.5 μg DNA per pair
- Water and NEB buffer aliquots, a few
- Part 3, purification
- loading dye aliquots
- 1% agarose gels
- TAE buffer
- Qiagen aliquots: DI water (100uL), QG (550uL), PE (1.8mL), EB (200uL), isopropanol
- Only put out as many columns as students should need
- Have psuedo-sterile eppendorf tubes out (were sterilized, but kept in open)
- Part 2, Western Day 2
- Plenty of TBS-T, in case folks over-wash
- Dilute 10x TBS in needed volume of water, reserving
- Space to dilute 10% tween (100X) to 0.1%
- Shaker area in fume hood cleaned up
- A few aliquots of GAR-AP
- I believe 24 mL distilled H2O aliquots were pre-prepped for them?
- Plenty of TBS-T, in case folks over-wash
Day of Lab (R/F):
- Set out ice buckets
- Prepare 50 °C heat block
- Place appropriate enzymes in orange freezer rack in alphabetical order
- Put out DNA ladder on cold rack as well
- Encourage students to pre-weight eppendorfs.
- Pipet-aids out for aliquotting 9 mL TBS-T
- Thaw/refrigerate development buffer if not already in fridge
- Briefly thaw reagents A and B and keep in the dark
- Measure 260 and 280 nm values for each pair's DNA, using Niles lab Nanodrop
After Lab:
- Take and post pictures of blots if students did not
How it went:
- Generally smooth, students in small section finished with ~20 min to spare.
- Next year, consider consistently running single cut DNA alongside the digests, and even separate out a bit longer to avoid single-cut contamination
Day 5
Materials required:
Day of Lab (T/W):
- Cell plating
- 1e5 K1 and 1.2e5 xrs6 per needed well in 24-well plate, plated morning of previous day
- probably can go a little lower in density for S15
- pre-treat half of K1 with inhibitor evening before lab
- 1e5 K1 and 1.2e5 xrs6 per needed well in 24-well plate, plated morning of previous day
- In advance: lots of sterile epps! One beaker per hood.
- One aliquot of each per team, including 20% excess or more
- OptiMEM
- LTX
- PLUS reagent
- Intact pMax-BFP(-ScaI)
- Intact pMax-GFP
- Extra cut DNA, plus their own thawed
After Lab:
Keep an eye on cell appearance, media color.
How it went:
WF left b/w 4 and 5 pm, depending on when they were in TC.
Day 6
Materials required:
- Part 1, flow cytometry
- several ice buckets
- purple tube holders (from 15 mL conicals) pre-chilled in fridge or on ice
- flow cytometry tubes with strainers, also pre-chilled
- pre-warmed PBS, phenol-red-free trypsin/EDTA, OptiMEM
- one aliquot per team
- well in advance: teaching faculty lipofections should be done same day as student ones
- single color controls, etc.
- Part 2, inhibitor dose response after irradiation
- in advance: plate one T25 per irradiation condition
- Compound 401 prepared in ethanol
Day of Lab (R/F):
- Pre-part 1
- TA collects teaching faculty samples
- Part 1
- TA brings ready samples to flow facility as needed
- Part 2
- might be done differently in S15
- TA/instructor trypsinizes cells, Samson lab person irradiates cells
- help students implement their plan smoothly
After Lab:
How it went:
Flow times were sufficient and students got out on time as well. For 6 groups, did 2:30-4:30; for 9 groups, did 2:30-5. Had plenty of time for analysis, etc. this way.
Day 7
Materials required:
Day of Lab (T/W):
After Lab:
How it went:
Day 8
Materials required:
Day of Lab (R/F):
After Lab:
How it went: