Problem: Highly concentrated nucleic acid and protein samples must be diluted before they can be read on most absorbance-based spectrophotometers, and conversely, diluted samples must be concentrated to within the spectrophotometer’s dynamic range before reading. In addition to the extended time needed for these sample corrections, if not precisely calculated and carefully performed, they can lead to incorrect sample results and skew downstream processes.
|Take3™ Trio Micro-Volume Plate from BioTek Instruments, Inc.|
|Take3 micro-volume analysis comparison between an in-situ assay and one conducted in a microtube using a 20:1 volume ratio of BCA working reagent to protein standard.|
Solution: Micro-volume quantification using an accessory such as BioTek’s Take3™ micro-volume plate (Figure 1), along with its complementary microplate spectrophotometers, reduces operator error while offering simple and efficient micro-volume readings. A nominal 0.5 mm optical pathlength obviates the need for sample dilution or concentration, and low 2 μL sample volumes conserve precious sample and reagents.
BioTek offers two versions of the Take3 micro-volume plate. The Take3 and Take3 Trio both use paired custom silica glass slides. The bottom slide(s) is coated with a hydrophobic stencil, forming 16 sample “microspots.” The Take3 plate has one sample slide, while the Take3 Trio bundles 3 slides to accommodate up to 48 uniform 2 μL samples and blanks to be read at one time. When the top plate(s) is lowered onto the droplets, they are sandwiched between top and bottom, creating a fixed vertical optical path. After measurement in the microplate reader, the aliquot may be removed and returned to the original vessel, or the device may be simply wiped clean as the surface generally resists binding.
Since multiple microspots may be measured at one time blanks, calibration curves and unknown samples may be simultaneously read for highly accurate results without variability from incubation time, temperature, and sample manipulation. Rapid measurements and results display are provided through preprogrammed protocols in the reader’s software. Colorimetric (Figure 2) and fluorometric measurements can be performed with Take3 plates, along with spectral scans and other micro-volume analysis.
In addition to microspot measurements, both accessories offer measurements via an optional, proprietary quartz vessel called BioCell. The BioCell offers a fixed, vertical 1 cm pathlength and uses less sample volume than traditional cuvettes. The pathlength is automatically normalized for each measurement type using the reader’s software so that results may be compared to those obtained via cuvette-based spectrophotometers.
Application bottlenecks and related sources of error due to concentrated or dilute samples can be avoided through the use of micro-volume quantification without sample dilution. Additionally, micro-volume quantification can reduce sample and reagents costs associated with most common absorbancebased molecular biology procedures and some fluorometric procedures.
For more information, visit www.biotek.com