| RLGS | 点击： 作者： 来源： 时间： 2007-04-10 本站论坛
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|  | Not I and EcoR V digestion
Mix the reaction solution mildly but completely with pipetting.
Centrifuge at 6,000 rpm for 1 min.
Incubate at 37oC for 3 hrs.
E. Labelling
Mix a 10 ul DNA sample with a 3.2 ul labelling solution gently with pipetting ten times.
Incubate at 27oC for 30 min.
Add 3 ul Stop solution.
Fill the cathodal tank (top) with 300 ml of 1x 1-D buffer.
Wash out the 1-D buffer containing 20% sucrose from the top of the gel.
Apply 10 ul (about 0.15 ug DNA for rice, or about 0.45 ug DNA for egg plant) sample on a 1-D gel.
Run electrophoresis at 130 Volts (120 - 140 Volts) for 40 hrs (until the center of BPB reaches 40 cm and that of XC reaches 19 cm from the top of the gel)(Photo) .
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.
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