| RLGS | 点击： 作者： 来源： 时间： 2007-04-10 本站论坛
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|  | 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.
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