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.

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 &mu;L 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 + &beta;-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 &mu;L
 * (1) 300ul aliquot of CII standard per group
 * prep 3.5 mL plus 35 &mu;L
 * 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 &mu;L 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)