4: GFP folding4: GFP folding margaret Wed, 11/02/2011 - 13:37
START UREA THE DAY BEFORE!
Hot blocks: set to 90C
MW = 154.25.
Frozen 1M stock - take out early, vortex. It looks like it will never go back into solution, but it will.
100 mM Tris, pH. 7.5, 150 mM NaCl, 10% glycerol, 5 mM DTT
students fill 12 wells @ 200 µL = 4800 µL, so aliquot 5 mL/group
Make 150 mL
UREA 9M + 1mM DTT
students need ~ 1537 µL/group. Aliquot 4mL/group
Make 125 mL
1M DTT: 0.125 mL
Urea MW = 60.06
vi = 9mol/L x 0.125L x 60.06g/mol = 67.568g
Stir overnight! Gets very cold! Add heat if necessary
Stir! Takes overnight! Gets very cold!
Do not refrigerate concentrated urea - it precipitates!
NOTE: 100 mL makes only just 25 aliquots of 4 mL!
Molar extinction coefficient of GFP = 8.33 x 104/M/cm
MW = 27000
A 4mg/mL solution = 1.48 x 10-4M
Stock solution in freezer is 9.6 mg/mL.
Students get 4 mg/mL GFP, then dilute it 10-fold. They need to dispense ~180 µL of the 0.4 mg/mL GFP.
Dilute 500µL 9.6 mg/mL stock with 700 µL TNG to make 1200 µL 4 mg/mL for student use:
Lab manual says to give them each 100 µL, even though they really need ~25 µL.
Make 50 µL aliquots, with extras available.
They make a 10-fold dilution with urea, to denature it. (They need about 200 µL final volume, 20 µL initial volume.)
They also make a 10-fold dilution with TNG for native GFP. (They need ~25 µL final volume, 2.5 µL initial volume.)