Bio-Rad Model 785 Vacuum Blotter - Manual (Page 11)

RNA Transfer Procedure:
1. After electrophoresis rinse the RNA gel in deionized distilled water.
2. Transfer in 10x SSC for 90 minutes at 5 inches of Hg. Follow the instructions in
Section 3.4 for the transfer procedures.
3.4 Vacuum Transfer
1. Wet the precut nylon membrane from Section 3.2 in double distilled water by slowly
lowering the membrane at a 45 degree angle to the water. Then, wet the membrane
and the filter paper in the appropriate transfer solution.
2. Make sure that the Porous Vacuum Plate lies flush on the Vacuum Stage. Place the
wetted filter paper on the Porous Vacuum Plate. Lay the filter paper in the area where
the cut window of the Gasket will be. Place the wetted membrane on top of the filter
paper. Remove the bubbles by rolling a 10 ml glass pipet over the membrane.
3. Wet the Reservoir Seal O-ring with water.
4. Place the Window Gasket on top of the membrane/filter paper. Make sure the
Window Gasket covers the entire O-ring on the Vacuum Stage. At the same time,
make sure that the filter/membrane is overlapping the Window Gasket. Realign as
necessary. Refer to Figure 2.
Alternatively, place the Window Gasket on the Porous Vacuum Plate first. Adjust
the Window Gasket to cover the entire O-ring. Then, hold one end of the Window
Gasket and slowly peel the other end back until the window area is up off the Porous
Vacuum Plate. Place the wet membrane/filter on the Porous Vacuum Plate under the
window area and under the Window Gasket. Finally, replace the partially peeled
back Window Gasket over the membrane/filter paper. Make sure the Window Gasket
covers the entire O-ring and overlaps the membrane/filter paper on all sides.
5. Gently place the gel, well side up, on top of the Window Gasket. The gel must
overlap the window. Remove bubbles by using a 10 ml glass pipet. As a final check,
make sure that the gel edges overlap the Window Gasket by at least 5 mm.
6. Place the Sealing Frame on top of the Vacuum Stage. Lock the Sealing Frame onto
the four latch posts. The spring latch handle of the Sealing Frame has a precut region
in the middle. Push down on this exposed area of the Sealing Frame with your thumb
until the latches snap onto all four latch posts.
7. Unscrew (counterclockwise) the Vacuum Regulator bleeder valve several turns to
prevent strong initial vacuum.
8. Start the vacuum source and slowly turn the bleeder valve clockwise until the gauge
reads at 5 in. of Hg. If the Bio-Rad Vacuum Pump is used, prewarm the pump for 10
minutes before blotting. Without prewarming, the vacuum pump will slowly increase
the pressure and then stabilize after 10 minutes. Adjust the pressure to 5 inches of Hg.
9. With a finger, apply gentle pressure on top of the gel along the window border. The
pressure helps to form a tight vacuum seal between the gel and the Window Gasket.
10. Gently pour 1,000–1,500 ml of the appropriate transfer solution (refer to Section 3.3)
into the upper reservoir. Check to see if the gel is displaced. The gel should stick to
the Window Gasket. If the gel floats, simply disassemble the Sealing Frame to drain
the transfer solution and repeat Steps 5 to 10 again.
11. Place the lid on top of the Vacuum Blotter. Occasionally check the buffer level. It
should be higher than the gel. Check the vacuum pressure and adjust the vacuum
pressure to 5 inches Hg as needed.
12. Transfer the gel for 90 minutes at 5 inches Hg.
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