Difference between revisions of "20.109(S14): TA notes for module 2"
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'''Materials required:''' | '''Materials required:''' | ||
− | Digests | + | *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? | |
− | + | ||
− | Dilute 10% tween (10X) | + | |
− | Dilute 10x TBS in total desired volume of water | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
+ | '''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:''' | '''After Lab:''' | ||
+ | |||
+ | *Take and post pictures of blots if students did not | ||
'''How it went:''' | '''How it went:''' | ||
− | Generally smooth, students in small section finished with ~20 min to spare. | + | |
+ | *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=== | ===Day 5=== |
Revision as of 16:17, 4 June 2014
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 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
- 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
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: