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

• Transfer buffer

• CHO cell media

Note that xrs6 and other Samson lab cell stocks will grow without NEAA. However, DAL lab K1 absolutely require NEAA, so during S14 it was used in all CHO media for consistency.

General cell upkeep