User Manual

Section 5

DNA Blotting

This section gives protocols for DNA blotting. The alkaline blotting method, using

Zeta-Probe

membrane, and the standard method for DNA blotting to nitrocellulose is

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.

2. Pre-wet 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; Zeta-Probe membranes should be wetted in

distilled water (see Section 10 for recipes).

3. Assemble the Bio-Dot 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

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 a Costar

Octapette pipet with the vacuum off and the flow valve open. Apply the denatured DNA in a

50–500 µl sample volume. Multiple loadings may be performed. However, best binding and

most rapid results occur using minimum sample volumes. Fill all wells with the same volume

to obtain homogeneous filtration.

5. The sample may be pulled through by applying a gentle vacuum, or by gravity filtration.

Notes: a method for applying gentle vacuum to the apparatus is to adjust the flow valve to

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 apparatus. Remove the blotted membrane and rinse it in 2x SSC.

Allow the membrane to airdry. 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

membrane 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.