Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 12)

Section 5
DNA Slot Blotting
This section gives protocols for DNA slot blotting. The alkaline blotting method, using Zeta-Probe
membrane, and a more standard method for DNA blotting to nitrocellulose, are described.
1. The target DNA must be denatured prior to application to the membrane. When using the
Zeta-Probe membrane, denature the DNA sample by addition of NaOH and EDTA solution to
final concentrations of 0.4 M NaOH, 10 mM EDTA. Heat the sample to 100°C for 10 minutes to
ensure complete denaturation. When applying DNA to a nitrocellulose membrane, denature the
DNA in the same manner. The DNA must then be neutralized by adding an equal volume of cold
2 M ammonium acetate, pH 7.0 to the target DNA solution. Leave DNA on ice while preparing
Bio-Dot.
2. Prewet the membrane by placing the membrane gently at a 45° angle into a tray of the
wetting solution. Always wear gloves when handling blotting membranes. Nitrocellulose
membranes should be wetted in 6x SSC (see Section 9 for recipes); Zeta-Probe membranes
should be wetted in distilled water.
3. Assemble the Bio-Dot SF apparatus according to the instructions in Section 3.1. Apply the
vacuum and then retighten the screws that hold the apparatus together. Rehydrate the
membrane with 500 µl Tris-EDTA (TE) or H
2
O as described in Section 3.1. At this point, the
unit is ready for sample application.
4. Samples and wash solutions should be applied with a standard pipet or an 8-channel pipet
with the vacuum off and the flow valve open. Apply the denatured DNA in a 50–500 µl
sample volume. The recommended sample loading volume is at least 200 µl. If sample
volumes of less than 200 µl are applied, they must be very carefully applied to the center of
the well. Applying the solution on one side of the well results in unequal distribution of DNA.
This may result in unevenly shaped bands, leading to distorted densitometer readings. Fill all
wells with the same volume to ensure homogeneous filration.
5. The sample may be pulled through by applying a gentle vacuum, or by gravity filtration.
Note: A method for applying gentle vacuum to the apparatus is to adjust the flow valve to
valve setting 3. Use a finger to cover the valve port exposed to air. The amount of vacuum
reaching the manifold will be regulated by the pressure of your finger on the valve.
6. After the sample has filtered through, add 500 µl 0.4 M NaOH to each well for Zeta-Probe
membrane, or 2x SSC for nitrocellulose. Apply the vacuum by setting the 3-way valve to
setting 1 until the sample wells are empty.
7. Disassemble the Bio-Dot SF apparatus. Remove the blotted membrane and rinse it in 2x SSC.
Allow the membrane to air dry. The Zeta-Probe membrane is ready for hybridization
immediately after air drying. If hybridization is not to be undertaken within 2 days, then
vacuum bake the blotted Zeta-Probe membrane at 80°C for 30 minutes. Nitrocellulose
membranes must be baked under vacuum for 2 hours at 80°C before hybridization. The
Zeta-Probe membrane and nitrocellulose membranes can be stored dry between two pieces
of filter paper in plastic bags at 23–25°C.
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