20.109(S16): TA notes for module 2

General notes

Scheme: Each pair of students will assay M059K and M059J cells for DNA-PKcs presence. They will then assess the ability of M059K, M059J and DNA-PKcs inhibited cells to repair DNA damage via plasmid based assay and flow cytometry.

Key preparation:

• A lot of cell culture in this module. At all times, both M059K and M059J need to be grown.
• Prepare cut DNA – one of each type – in advance, in case student yields are low.
• Large preps of uncut endotoxin-free DNA can readily be purchased from Genewiz.
• If MCS design is to be changed, 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.

Day-by-day

Day 1

Materials required:

All for Part 2, cell plating for Western blot.

• Two T25s per group, one of M059J and one of M059J.
  • Flasks for day after next, seeded at 200,000 cells
• One shared aliquot per team
  • PBS, media, and 0.05% trypsin
• A few aliquots of Trypan Blue at the scopes
• Equipment to have out
  • 6-well plates

Day of lab:

• Partially prepare TC hoods (vacuum lines, bleach, stock pipets, autoclaved eppendorf tubes).
• Warm up media, PBS, and trypsin half hour before students use reagents.
• During class, assist students.

Day 2

Materials required:

• Part 1, cell lysate preparation
  • ice bucket at each bench, with
    • four empty eppendorf tubes
    • RIPA buffer (exactly 250 uL)
    • protease inhibitors (2.5 uL) – distributed by instructor at last minute
    • PBS (~4 mL)
• Part 2, protein measurement and preparation
  • cuvettes at front bench
  • Precision Red reagent aliquots, 3mL
  • water for diluting lysates
• Part 3, SDS-PAGE
  • ice bucket on front bench with Kaleidoscope ladder and Laemmli aliquots
  • inside fume hood:
    • boil water with chips at about level 5
    • prepare caps for boiling tubes
  • at gel bench:
    • 4L of well-mixed TGS prepped in advance
    • gel chambers set up – make sure to remove strip from bottom!
• Part 4, transfer step
  • in advance: freeze ice packs
  • cassettes, pads, filter paper
  • membranes, scissors at a cutting station
  • green gel openers
  • 4L transfer buffer
  • blocking buffer (Odyssey)

Day of lab:

• 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: train students at hood (caps) and SDS-PAGE (how and where to load samples)
• Part 4:
  • pre-soak ScotchBrite pads in cold transfer buffer
  • explain transfer in advance to keep things moving

After lab:

Move their blots to blocking buffer.

Day 3

Materials required:

• Part 1, digests
  • Prepare on instructors' bench:
    • filtered tips
    • 7 μg DNA per group
    • water and NEB buffers
    • restriction enzymes: PmeI, EcoRI, BglII, and PstI
• Part 3, purification
  • loading dye aliquots
  • 1% agarose gels (3 teams per gel)
  • TAE buffer (2L)
  • 50 °C water bath (to dissolve agarose in QG)
  • Qiagen aliquots (per team): QG (1.5 mL), isopropanol (250 μL), PE (900 μL), and water pH 8.0 (50 μL)
  • Only put out as many columns as students should need
  • Have pseudo-sterile eppendorf tubes out (were sterilized, but kept in open)
• Part 2, Western Day 2
  • TBS-T (2L)
    • Dilute 10x TBS in needed volume of water, reserving
    • Space to dilute 10% tween (100X) to 0.1%
  • aluminum foil
  • Saran wrap to preserve blots at -20 °C

Day of lab (R/F):

• Place appropriate enzymes in orange freezer rack in alphabetical order
• Put out DNA ladder on cold rack as well
• Encourage students to pre-weigh eppendorfs.
• Pipet-aids out for aliquotting 9 mL TBS-T

After Lab:

• Take and post pictures of blots if students did not

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===