User Manual

4. Always use forceps or wear gloves when handling membranes. Prewet the nitrocellulose

or Zeta-Probe

membrane by slowly sliding it at a 45° angle into wetting solution. Note:

PVDF membrane is not recommended. Wet nitrocellulose in 6x sodium, sodium citrate

(SSC) for nucleic acid applications, and in tris-buffered saline (TBS) for protein blotting.

Wet Zeta-Probe membrane in distilled water. See Sections 9 and 10 for solution

preparation. A 10 minute soak is recommended for complete wetting of the membrane

to insure proper drainage of solutions. Remove the membrane from the wetting solution.

Let the excess liquid drain from the membrane. (Touching the membrane to a sheet of

filter paper is a simple method for removing excess buffer.) Lay the membrane on the

gasket in the apparatus so that it covers all of the holes. The membrane should not

extend beyond the edge of the gasket after the Bio-Dot apparatus is assembled.

Remove any air bubbles trapped between the membrane and the gasket.

Note: PVDF membrane is not recommended.

5. Place the sample template on top of the membrane. The guide pins ensure that the

template will be properly aligned. Finger–tighten the four screws. When tightening the

screws, use a diagonal crossing pattern to ensure uniform application of pressure on

the membrane surface (see Figure 2).

Fig. 2. Diagonal crossing pattern for tightening screws in the Bio-Dot apparatus.

6. Attach a vacuum source (house vacuum or a vacuum pump) to the flow valve with a

waste trap set up and positioned between the vacuum outlet and the flow valve. Turn

on the vacuum and set the 3-way valve to apply vacuum to the apparatus (flow valve

setting 1, Figure 3).

7. With vacuum applied, repeat the tightening process using the diagonal crossing pattern.

Tightening while vacuum is applied ensures a tight seal, preventing cross contamination

between slots. Failure to tighten screws during application of vacuum prior to

starting the assay may lead to leaking between the wells.

8. Adjust the flow valve so that the vacuum manifold is open to air pressure (flow valve

setting 2, Figure 3). Apply 100 µl buffer to all 96 sample wells. Use of the 8-channel

pipet and buffer reservoirs (see Section 13 for ordering information) will simplify the

process of adding solutions to the Bio-Dot apparatus. Addition of buffer is necessary

to rehydrate the membrane following the vacuum procedure in step 7. If this step is

not performed prior to applying samples, assay results will show halos or weak

detection signal.

9. Gently remove the buffer from the wells by vacuum (flow valve setting 3, Figure 3).

Watch the sample wells. As soon as the buffer solution drains from all the wells,

adjust the flow valve so that the unit is exposed to air and disconnect the vacuum. At

this point, the unit is ready for sample application.