20.109(S09): TA notes for module 3

General notes

Key preparation:

• Chondrocytes
• MSCs

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

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)