20.109(S09): TA notes for module 3

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General notes

Key preparation:

  • Chondrocytes
  • MSCs

Scheme: Each pair of students will make two cultures, with various modifications as proposed on Day 1.

Chondrocyte and MSC isolation protocol

Daily Notes

Day 1

No Quiz

Materials required:

  1. None: all work today is computer work. Get passing familiarity with the faculty-selected journal articles.

Day 2

Materials required:

  • Make sure to go through each group's plan to know which factors will be modified.
  1. 150 mM NaCl in autoclaved water--needs to be sterile filtered
  2. 102 mN CaCl2 in autoclaved water--needs to be sterile filtered
  3. Cell culture media
  4. Alginate--needs to be made the day before lab, allowed to dissolve at 4C overnight, then sterile filtered

Day of Lab:

  • Quiz (prepared by TA)
  • Need (2) 20ml aliquots of CaCl2 per group
  • Need (4) 20ml aliquots of NaCl per group
  • Need 2ml alginate per group

Day 3

Materials required:

  1. HBSS
  2. dye solution in HBSS
  3. 4% GAH in HBSS
  4. 1 sterile spatula per group
  5. 1 50ml conical tube for waste per hood
  6. 2 Petri dishes per group
  7. slides and coverslips

Day of Lab:

  • No Quiz

Day 4

Materials required:

  • Cell prep
    • lots of empty eppendorfs (don't need to be sterile)
    • waste tubes or beakers, for aspirations with ser. pipets
    • EDTA-citrate buffer (6 mL per group)
    • complete(ish) medium (9 mL per group)
    • a few eppendorfs w/100 μL of Trypan aliquotted
    • 4 eppendorfs of each of the following per day: 0.6 mL EDTA-citrate, 0.2 mL acetic acid, 0.2 mL pepsin
  • RNA prep and RT-PCR
    • 5 ice buckets
    • Water aliquots for spec. measurement
    • Thaw RT-PCR reagents (esp. water) early
    • RLT + β-merc last minute
    • Prep Master Mixes last minute
    • Turn on spec. last minute

Day of Lab:

  • Quiz
  • See "last minute" above
  • Note that g = rcf

Day 5

Materials required:

  • ELISA Day 1
    • (2) 96-well plates per group
    • (1) 300ul aliquot of CI standard per group
      • prep 3.5 mL plus 35 μL
    • (1) 300ul aliquot of CII standard per group
      • prep 3.5 mL plus 35 μL
    • PBS for diluting standards
    • eppendorfs
    • wash buffer
    • block buffer
    • primary antibodies at 1:4000
      • total needed per antibody ~ 25 mL
      • 3 batches of 10 mL PBS + 2.5 μL antibody
    • parafilm
  • Agarose gel
    • loading dye
    • sterile water
    • (2) 1.2% gels per day

Day of Lab:

  • Quiz
  • RT-PCR products out on cold block
  • protein samples briefly thawed just before lab

Day 6

Materials required:

  • ELISA Day 2
    • secondary antibody (1:1000, prep 10-15 mL at a time, in block buffer)
    • wash buffer
    • development buffer (1ml of development in 4ml of water per group)
    • pNPP (p-nitrophenyl phosphate) pellets (1 per 5 mL buffer, i.e., per group)
    • aluminum foil
    • stop solution (0.4 M NaOH)

Day of Lab:

  • Quiz
    • couple of multichannels at the sink, couple up front
    • students add antibody, development solutions at front bench, sharing 2 dishes per soln.

Day 7

Materials required: None--all computer work today

Day of Lab:

  • Quiz (final one)