* Use Positive displacement pipette tips throughout*

1) Make Collagen II 2mg/ml with 10mM Acetic Acid

2) Dialyze Collagen against 1000 volumes 10mM Acetic Acid 4° o/n

3) Dialyze CMP into 2x F.B. at 4°C

4) Filter sterilize and degas 15 ml of Milli-Q and 2x F.B.

5) Mix ingredients in 2ml tube on Ice in this order.
Total Volume is 250µl. Total Concentration of F.B. is 1x
Water
2x F.B.
CMP in 2x F.B.
Collagen II (mix with repeated pipetting immediately)

* Turn UV source OFF for fibrillogenesis *
* Blank spectrophotometer on water in first cuvette *
* Match all cells with water *

6) With 250µl Pipetman, add mixture to 37°C cuvette. Water bath must be set to 38.1°C for cuvette to be 37°C on average.

8) Print results, do not save, because machine does not have enough memory.

*Mineral Oil may be layered over reaction, but interferes with EM.*

Type II collagen conditions:
-200µg/ml collagen, CMP to equimolar concentrations
-2x FB is 120mM NaCl, 60mM Sodium Phosphate pH 7.30
(76.5% dibasic, 23.5% monobasic) then adjust to 7.30

Type I collagen conditions:
-600µg/ml collagen, CMP to equimolar concentrations
-2x F.B. is 280mM NaCl, 60mM Sodium Phosphate pH 7.30
(76.5% dibasic, 23.5% monobasic) then adjust to 7.30