20.109(S14): TA notes for module 2
General notes
I need to figure out the quiz dates.
See also GoogleDoc 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 to repair DNA damage via plasmid based assay and flow cytometry.
Key preparation:
Cells:
- Prepare T25 flasks of K1 and xrs6 for every team
Westerns:
- Prepare transfer buffer
DNA plasmids:
- Aliquot plasmids
Lipofection: Flow cytometry:
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 GoogleDoc as needed.
Day-by-day
Day 1
Materials required:
1. Aliquots of PBS, CHO media, and 0.25% Phenol Red Trypsin for each group 2. T25 flask of K1 and xrs6 cells for each group
- 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
Day of Lab (T/W):
- No Quiz
- Prepare remaining aliquots for students
- Warm up media, PBS, and trypsin half hour before students use reagents
Additional Prep.:
- Digest pMAX-BFP-2MCS2 for testing damage topologies
- Pour gel for purification
After Lab:
- Aliquot new shipment of trypsin
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 and second day of Westerns
Thaw buffers, make aliquots place appropriate enzymes in orange rack Make aliquots of tubes, water, QG, PE, EB, isopropanol, spin columns Pour gels Make TE buffer Make enough ladder Prepare control digest, if desired Set out ice buckets
Western prep: Aliquots if 2ndary Ab Thaw development buffer / reagents A and B Make chilled TBS-T (~2L) Recipe: Dilute 10% tween (10X) Dilute 10x tbs In total desired volume of water
Day of Lab (R/F):
After Lab:
How it went: Generally smooth, students in small section finished with ~20 min to spare.
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:
