DNase (2X) and papain (2X)
DNase and papain from 2X
This is based off of Worthington Papain Dissociation System to achieve a final ** 20 U papain/mL + 2,000 U DNase I/mL solution with 0.5mM EDTA and 1mM L-cysteine**.
Add equal volumes 2X DNase and papain from the -80°C. e.g. 1 mL DNase (2X) + 1 mL papain (2x).
Solution |
Details |
|---|---|
4,000 U/mL in DMEM/F12 |
|
40 U/mL with 1mM EDTA, 2mM L-cysteine in DMEM/F12 |
L-Cysteine (50 mM)
Measure 60.6 mg L-Cysteine (MW: 121.16 g/mol) and add to a 15 mL conical
Add 10 mL DMEM/F12
Take into TC and sterilize by filtering through a 0.22 µm filter
Aliquot ~1.1 mL into 1.6 mL eppendorf tubes and freeze with papain aliquots at -80°C
Papain (2X: 1mM EDTA, 2mM L-cysteine, 40 U papain/mL)
To make a 50 mL solution of 2X papain from Papain 100 mg:
Component |
Final conc |
Volume |
|---|---|---|
EDTA (0.5M, pH=8.0) |
1 mM |
100 µL |
L-Cysteine (50mM) |
2 mM |
2 mL |
Papain (100 mg bottle) |
40 U/mL |
100 µL |
DMEM/F12 |
47.9 mL |
Sterilize with 0.22 µM filter and store aliquots at -80°C. EDTA (0.5M, pH=8.0) should be in Bruni and L-Cystein (50mM) should be in -80°C with papain aliquots.
DNase (2X: 4,000 U/mL solution)
To make a 2X solution from DNase I (100 mg, 2000 units/mg).
Add 50 mL DMEM/F12 to a 100 mg bottle, sterilize with 0.22 µM filter and store aliquots at -80°C.