4: GFP folding

START UREA THE DAY BEFORE!

Hot blocks: set to 90C


DTT 1M

MW = 154.25.

Frozen 1M stock - take out early, vortex. It looks like it will never go back into solution, but it will.

To make mL 1M DTT
add: g DTT (dry, stored in freezer)


TNG
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

To make mL TNG
add: mL 1M Tris pH 7.5
add: mL 5M NaCl
add: mL glycerol
add: mL 1M DTT


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

To make mL Urea-DTT
add: g urea
add: mL H20
add: mL 1M DTT

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!


GFP
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.)

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lab_4-instructor.doc61 KB