Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus User Manual
Page 23
Section 10
Troubleshooting Guide
I. Filter Apparatus
1. Leakage or cross-well contamination
a.
Improper assembly. The screws must be retightened under vacuum following the initial
assembly. The thickness of filter paper must be correct, or leakage will result. Exactly
three sheets of filter paper must be placed on the membrane support. Do not use filter
paper other than the Bio-Dot SF filter paper.
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. Pipet liquid in the wells up and down to displace
bubbles. Use a needle to break any bubbles, being careful not to puncture the
membrane.
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.
3. Halos
a.
Membrane is not properly rehydrated before applying samples. Always rehydrate
membrane prior to applying any sample.
b.
Use of more than three filter paper sheets or more than one membrane will cause lateral
diffusion and leakage.
c.
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 Zeta-Probe membrane as an alternative.
c.
Mixed-ester cellulose binds DNA, RNA, and protein very poorly. Use Bio-Rad’s pure
nitrocellulose.
d.
Proteins—15,000 daltons may show diminished binding to 0.45 µm nitrocellulose. Use
the Zeta-Probe membrane or 0.2 µm nitrocellulose. Also, glutaraldehyde fixation will
increase retention of small proteins and peptides to both nitrocellulose and Zeta-Probe
membrane.
f.
Protein may be removed from nitrocellulose by SDS, NP-40, Triton X-100. Use Tween 20
in washes. Reduce concentrations or time of any SDS or NP–40 washes.
19