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: Please read as much as you can of Parts 1-3 and create an automated worksheet (e.g., in Excel) that will perform the required calculations for the day. A partially prepared worksheet is linked here. We will NOT collect your final worksheet this time; it is only for your benefit.

Prep. For westerns: Make aliquots: Chill scrapers RIPA lysis buffer Protease inhibitor (last minute) Eppendorf tubes PBS for wash Ice buckets

Set up boiling station: Turn on hot plates with water in glass bowl with boiling chips Aliquot beta-mercap. Don't forget to show students the cap covers

Set up westerns: Make sds running buffer Make transfer buffer (chilled) Set up gels(remove strips, place in boxes, remove combs, wash wells with running buffer) Night before: freeze ice blocks

Presoak filter paper and scotch pads in transfer buffer for 30 min prior to blot.

Make sure we have blocking buffer.

Day of Lab (R/F):

How it went:

Day 3

Materials required: None, your brains :)

Day of Lab (T/W): First quiz

After Lab:

How it went:

Day 4

Materials required:

• Digests
  • Aliquot exactly 3.5 μg DNA per pair
  • Water and NEB buffer aliquots, a few
• 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/once-sterile eppendorf tubes out
• Western Day 2
  • Plenty of TBS-T, in case folks over-wash
    • ?Dilute 10% tween (10X)
    • ?Dilute 10x TBS in total desired volume of water
  • 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

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):

After Lab:

How it went:

Day 6

Materials required:

Day of Lab (R/F):

After Lab:

How it went:

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: