User Manual

Section 10

Troubleshooting Guide

I. Filtration Apparatus

1. Leakage or Cross-Well Contamination

a. Improper assembly. The screws must be retightened under vacuum following the

initial assembly.

b. Membrane is not properly rehydrated after assembly. Always rehydrate the

membrane prior to applying samples. Apply vacuum only until solutions are

removed from the sample wells, then disconnect the vacuum source.

2. No Filtration or Uneven Filtration Occurring

a. Macromolecular polymers, cellular debris, or dirt is plugging the membrane.

Centrifuge samples prior to application to remove particulates. Filter solution prior

to use to ensure removal of particulate material. Cover wells with Parafilm during

lengthy incubations.

b. Bubbles are obstructing the filtration. Use a needle to break any bubbles, being

careful not to puncture the membrane. Pipet liquid in the wells up and down to

displace bubbles.

c. The flow valve is positioned higher than the apparatus. The flow valve must be

lower than the level of the sample wells on the apparatus for proper drainage to

occur.

d. Improper blocking reagent is used. BSA is the recommended blocker for

nitrocellulose; gelatin will plug the apparatus, and no filtration will occur. The

Zeta-Probe

membrane, which requires more stringent blocking with BLOTTO or

with gelatin and MPO, should be removed from the Bio-Dot apparatus following

antigen immobilization and the rest of the assay should be conducted in a

separate container.

3. Halos

a. Membrane is not properly rehydrated before applying samples. Always rehydrate

membrane prior to applying any sample.

b. Excessive concentrations of sample are loaded. When too much sample is

present, wicking into the membrane around the well will occur. Use serial dilutions

of the samples to determine optimal amounts to load.

II. Poor Binding to Membrane

1. Nitrocellulose

a. DNA/RNA will only bind efficiently in 20x SSC or 1 M ammonium acetate. Use the

Zeta-Probe membrane as an alternative.

b. DNA must be single stranded and RNA must be denatured. DNA 500 bp may

not bind to nitrocellulose. Use the Zeta-Probe membrane as an alternative.

c. Mixed-ester cellulose binds DNA, RNA, and protein very poorly. Use Bio-Rad’s

pure nitrocellulose.