20.109(S14): TA notes for module 2

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?

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
• 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:

All for Part 1, colony staining.

• PBS, room temperature. (First step can be pre-warmed to 37 °C but not essential.)
• Biosafe Coomassie aliquots (>12 mL) per team, serological pipets and pipet-aid up front

Day of Lab (T/W):

Be prepared to assist with flow cytometry data analysis.

After Lab:

Compile student data into one master worksheet and share.

How it went:

Most WF folks were leaving around 4, some as early as 3:15.

Buffer and media compositions

General cell upkeep