F. Preparation of 2-D Gel
Siliconize one side (the side with a tapered edge) of a glass plate.
Set up the gel casting apparatus (Bio-craft).
Mix a gel solution and degas it by vacuum with a sonicator for 5 min.
Cover the top of the gel with Kim Towel wetted by 1 x TBE containing 1% glycerin to keep wet.
G. in situ digestion
After electrophoresis remove the cathodal top buffer with aspirator and take out each gel holder.
Expel the gel slowly using a shortened yellow tip on a 1 ml syringe filled with 0.05% BPB water 10x high buffer (HB)(Photo)
Cut off the top 15 cm of the gel at 45o .
Soak the gel in 50 ml tube containing 40 ml of 1x HB.
Shake the tube gently and equilibrate the gel for 10 min.
Change the buffer and equilibrate the gel once again for 10 min.
Pour the equilibrated gel into a stainless tray.
Cut off lower 12 cm of the gel at 90o.
Place the gel noodle (34 cm long corresponding with 500 bp to 7 kb) in a plastic tray of 22(W) x 350(L) x 3(D) mm.
Remove any trace of the buffer.
Pour 6 ml of 1x HB containing 1600 units of MboI and 0.01% BSA (enzyme solution).
Seal the tray with Saran Wrap (incubate at 37oC for 2 hrs set up the electrophoresis apparatus (Bio-craft).
Fill the electrode tanks with 1x TBE buffer (3 L for the bottom tank).
H. Running 2-D gel electrophoresis
Rinse the top of the 2-D gel with 1x TBE and wipe with Kimwipe after in-situ digestion with MboI expel the gel noodle from tubing into a 50 ml tube containing 40 ml of 1x TBE.
Equilibrate for 10 min.
Discard the buffer and pour the gel noodle onto a plastic board.
Transfer the gel onto the top of 2-D gel.
Connect agarose noodle gel and 2-D gel with 3 ml of connection agarose gel solution using a 5 ml syringe with 20 G needle (Photo).
Overlay dye agarose gel solution using a 5 ml syringe with 20 G needle.
Fill the electrorode tank with 1x TBE buffer (1.5 L for the upper tank).
Run electrophoresis at 135 Volts (130 - 140 Volts) for 40 hrs until the XC reaches about 33 cm from the top (BPB indicates about 65 bp and XC indicates about 260 bp).
I. Gel drying
Cover the gel with a piece of filter paper (3 MM, 345 mm x 425 mm).
Cut off the gel outside of the filter paper (Photo).
Take up the gel with the filter paper, and lay it down on two pieces of filter paper (3 MM, 360 mm x 440 mm) with the gel side up.
Overlay Saran Wrap (45 cm wide) onto the gel, and then a piece of filter paper (3 MM, 360 mm x 440 mm) on them.
Cut off an excessive area of the Saran Wrap remaining 5 mm edge.
Set the gel on a gel dryer (Bio-Rad, Model 583).
Dry up the gel at 60oC for 1.2 hr.
J. Autoradiography
Set an intensifying screen (on the bottom), the dried gel, and then a piece of filter paper (3M) on them in a film cassette.
Insert an X-ray film (KODAK X-OMAT AR) between the gel and the intensifying screen in a dark room.
Perform autoradiography at -80 oC for 60-64 hr.
Warm the cassette up to the ambient temperature.
Develop, fix and dry the X-ray film in a dark room. 上一页 [1] [2] [3]
