Biotek Synergy H1, Operator'S Manual

Page: 65 / 190

Recommendations for Optimum Performance
General
Microplates should be clean and free from dust or bottom scratches. Use new
microplates from sealed packages. Do not allow dust to settle on the surface of the
solution; use microplate covers or seals when not reading the plate. Filter solutions to
remove particulates that could cause erroneous readings.
Before preparing your microplates, make sure the reader is on and communicating with
Gen5. You may want to run a System Test if the reader has not been turned off/on in a
few days. Design your Gen5 protocol in advance as well, to ensure that the intended
reading parameters are used and to avoid any last-minute corrections.
Although the Synergy H1 supports standard flat, U-bottom, and V-bottom microplates,
the reader achieves optimum performance with flat-bottomed wells when running in
Absorbance mode. See
Specifications starting on page 139 for more information on
the supported plates.
Non-uniformity in the optical density of the well bottoms can cause loss of accuracy,
especially with U- and V-bottom polyvinyl microplates. Check for this by reading an
empty microplate. Dual wavelength readings can eliminate this problem, or bring the
variation in density readings to within acceptable limits for most measurements.
Inaccuracy in pipetting has a large effect on measurements, especially if smaller
volumes of liquid are used. For best results in most cases, use at least 100 µL per well
in a 96-well plate, 25 µL in a 384-well plate, and 5 µL in a 1536-well plate (if supported).
Pipetting solution into 384- [and greater] well plates often traps air bubbles in the wells,
which may result in inaccurate readings. A dual-wavelength reading method usually
eliminates these inaccuracies. For best results, however, remove the air bubbles by
degassing the plate in a vacuum chamber or spinning the plate in a centrifuge before
reading.
The inclination of the meniscus can cause loss of accuracy in some solutions, especially
with small volumes. Shake the microplate before reading to help bring it within
acceptable limits. Use Tween 20, if possible (or some other wetting agent) to normalize
the meniscus for absorbance measurements. Some solutions develop menisci over a
period of several minutes. This effect varies with the brand of microplate and the
solution composition. As the center of the meniscus drops and shortens the light path,
the density readings change. The meniscus shape will stabilize over time.
Use of liquids with concentrations of acids, corrosives, or solvents of 3% and greater
can begin attacking the materials inside the instrument’s chamber. Running multiple
plates with concentrations <3% in long kinetics may also have a destructive effect. If
the experiment is incubated, deterioration of chamber components will be accelerated.
When in doubt about the use of acids, corrosives, or solvents; please contact
TAC@biotek.com.
Synergy H1 Operator's Manual
Recommendations for Optimum Performance | 39