20.109(S14): TA notes for module 2

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Day 1)
(Day 2)
Line 85: Line 85:
'''Day of Lab (R/F):'''
'''Day of Lab (R/F):'''
 +
 +
'''After Lab:'''
 +
 +
Move their blots to blocking buffer.
'''How it went:'''
'''How it went:'''
 +
 +
W/F students out b/w 4:25 and 5:15 (most at 5 pm). Blocking transfer completed by 6:45 pm.
===Day 3===
===Day 3===

Revision as of 14:52, 17 June 2014

20.109(S14): Laboratory Fundamentals of Biological Engineering

Home        Schedule Spring 2014        Assignments       
Module 1        Module 2        Module 3              

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):

After Lab:

Move their blots to blocking buffer.

How it went:

W/F students out b/w 4:25 and 5:15 (most at 5 pm). Blocking transfer completed by 6:45 pm.

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?

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:
Personal tools