20.109(S14): TA notes for module 2
Contents
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?
- 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
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
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After Lab:
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Day 7
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Day of Lab (T/W):
After Lab:
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Day 8
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Day of Lab (R/F):
After Lab:
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