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 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 Dropbox Excel doc 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

Used Qiagen kit:
Make aliquots of tubes (2, pseudo-sterile), DI water (100uL), QG (550uL), PE (1.8mL), EB (200uL), isopropanol, spin columns
Pour gels
Make TE buffer
Make enough ladder (1kb)
Prepare control digest, if desired
Set out ice buckets

Western prep:
Aliquots of secondary Ab (goat and-rabbit alkaline phosphate conjugate)
Thaw development buffer / reagents A and B
Make chilled TBS-T (~2L total)
Recipe: Dilute 10% tween (10X)
Dilute 10x TBS in total desired volume of water


Day of Lab (R/F):
Set out water bath/heat block (50C)
Prepare 6xNEB loading dye stations for when students' digests are ready
Start weighing station for eppendorf tubes 10 minutes before gel is set to finish.

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: