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Suggs Lab



Mammalian Expression System, pTarget Promega



Ready Gel Precast Gels
1. Ready Gel Cassette Preparation
Note: The Mini-PROTEAN 3 cell is guaranteed for use only with Bio-Rad's Ready Gel precast gels.
  a. Remove the Ready Gel from the storage pouch.
  b. Gently remove the comb and rinse the wells thoroughly with distilled water or running buffer.
  c. Cut along the dotted line at the bottom of the Ready Gel Cassette with a razor blade.
  d. Pull the clear tape at the bottom of the Ready Gel Cassette to expose the bottom edge of the gel.
  e. Repeat for second Ready Gel.
Note: If only one gel is to be run, use the mini cell buffer dam.

Mini-PROTEAN 3 Electrophoresis Module Assembly 1. Remove the Gel Cassette Assemblies from Casting Stand. Rotate the cams of the Casting Frames inward to release Gel Cassette Sandwich. 2. Place Gel Cassette Sandwich into the slots at bottom of each side of the Electrode Assembly. Note: Short Plate of the Gel Cassette Sandwich faces inward toward notches of U-shaped gaskets. 3. Lift Gel Cassette Sandwich into place against green gaskets , slide into the Clamping Frame. 4. Press down on Electrode Assembly while closing cam levers of the Clamping Frame to form the Inner Chamber and to insure a proper seal of the short plate against the notch on the U-shaped gasket. Short plate must align with notch in gasket. 5. Lower the Inner ChamberAssembly into the Mini Tank. Fill the inner chamberwith ~125 ml of running buffer (reaches 1/2 between tops of the taller and shorter glass plates of the Gel Cassettes). Note: Do not overfill the Inner Chamber Assembly. Excess buffer will cause the siphoning of buffer into the lower chamber which can result in buffer loss and interruption of electrophoresis. 6. Add ~200 ml of running buffer to the Mini Tank (lower buffer chamber).

Power Conditions 1. Insert the electrical leads into a suitable power supply with the proper polarity. 2. Apply power to the Mini-PROTEAN 3 cell and begin electrophoresis; 200 volts constant is recommended for SDS-PAGE and most native gel applications. Run time is approximately 35 minutes at 200 volts for SDS-PAGE.


MSC media preparation

Item [Stock] [Media] Amt/500mL Store
Dexamethasone .25 nM .05 nM 100 uL (-80)
DMEM 1X .6X 300 mL (4)
EGF 10 ug/mL 10 ng/mL 500 uL (-80)
FCS 100% 1% 10 mL (-20)
ITS 100% 1% 5 mL (4)
LA-BSA 100 mg/mL 1 mg/mL 5 mL (4)
L-ascorbic acid salt 10 nM .1 nM 5 mL (-20)
L-glutamine 200 nM 2 nM 5 mL (-20)
MCDB 201 Medium 1X .4X 200 mL (4)
Pen/strep 100X 1X 5 mL (-20)
PDGF 10 ug/mL 10 ng/mL 500 uL (-80)

Filter at end.

MSC subculturing

MSC freezing