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:

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Day 4

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Day of Lab (R/F):

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Day 5

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Day 6

Materials required:

Day of Lab (R/F):

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Day 7

Materials required:

Day of Lab (T/W):

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Day 8

Materials required:

Day of Lab (R/F):

After Lab:

How it went: