Bio Research International BRI February 2011 : Page 10

Bio Resear ch Inter national to view this issue in interactive digital magazine format visit to view this issue in interactive for digital latest magazine news updates format visit visit www.BiotechDaily.com www.LinkXpress.com Micro-Volume Nucleic Acid Determinations with a Microplate Accessory by Peter J. Brescia, Jr., MSc, Applications Scientist, BioTek Instruments, Inc. Figure 1 A N ucleic acid quantification and purity assess-ment are common, yet critical, methods in molecular biology. Typically, spectrophotometric measurements made at wavelengths of 260 nm and 280 nm provide concentration and purity informa-tion -both essential to downstream application requirements for PCR, RT-PCR, qPCR, sequencing, restriction digestions, ligations etc. Spectrophotometric nucleic acid determinations have traditionally been made in glass, quartz or UV-transparent cuvettes with a fixed, horizontal pathlength of 1 cm. Low volume cuvettes are also available typically with a 1.0 cm pathlength. Spectrophotometric cuvettes typically require vol-umes of 0.5 to 3.5 mL. The fixed horizontal path-lengths of traditional cuvettes allow single rapid determination of concentration. Microplate spectrophotometers and UV-transpar-ent microplates now provide increased throughput with the capability for rapid measurement of 96 or 384 samples, with volumes from 10 to 350 µL. In a microplate, the sample pathlength is vertical, and varies based on the volume used. Pathlength nor-malization to a 1 cm standard is required for rapid quantification of microplate based measurements to compensate for volume variations, in accordance with the Beer-Lambert Law. It is often necessary to dilute samples for measurement in a microplate or cuvette, as the absorptivity of nucleic acids at 260 nm is high. A new alternative to cuvette-or microplate-based nucleic acid and protein determinations is a micro-volume device such as the Take3!" Multi-Volume Plate (BioTek Instruments, Inc). The Take3 plate allows up to sixteen 2 µ L samples to be meas-ured quickly and easily in a microplate spectropho-tometer. The Take3 plate also can measure sample in a cuvette or in BioTek s patented 1 cm BioCell!" and is suitable for colorimetric and fluorescent measurements in a microplate reader. The micro-volume capability of the Take3 plate allows for conservation of precious sample and eliminates the need for dilution. The Take3 micro-spots have a nominal 0.5 mm fixed vertical path-length. The Take3 uses two non-boronated glass slides that generally resist non-specific binding. The bottom slide of the Take3 plate is coated with a hydrophobic surface in a 2 x 8 pattern containing 2 mm sample micro-spots . The hydrophobic coating facilitates proper sample droplet formation; when the cover of the plate is closed, the sample droplet is sandwiched between, forming the nominal 0.5 mm pathlength for each sample. After measure-ment, the sample can be removed and returned to its original vessel, or it may be simple wiped clean. The short 0.5 mm pathlength provides increased accuracy particularly at upper detection limits com-pared to that of a 1 cm pathlength, so nucleic acid and protein samples do not require dilution to fall within narrow detection limits. For colorimetric methods, the standard curve and unknown sample data may be generated at the same time, reducing variability from incubation time, temperature and sample manipulation. Comparing Take3 data to 1 cm pathlength methods Take3 micro-volume performance was compared to BioCell cuvette performance using an Epoch!" Microplate Spectrophotometer. This comparison serves to confirm that Take3 micro-spot measure-ments are equivalent to traditional 1 cm pathlength measurement methods. The unique quartz BioCell allows for absorbance readings with a fixed 1 cm vertical pathlength in BioTek s microplate readers. The monochromator-based Epoch Microplate Spectrophotometer meas-ures absorbance from 200-999 nm in 6-to 384-well microplates, the Take3 Multi-Volume plate, and the BioCell. Along with endpoint readings for nucleic acid and protein quantification, Epoch can also per-form spectral scanning, kinetic and well area scan-ning measurements. Concentrated herring sperm double-stranded DNA (dsDNA) in TE buffer (10 mm TRIS, 1 mm EDTA, pH=7.0) was serially diluted to create a series of standards with a concentration range over three orders of magnitude. Two microliters of each undiluted standard concentration was loaded five times at each Take3 micro-spot location using an 8-channel manual pipette. Undiluted standard con-centrations or standards diluted 20-fold in TE buffer, were loaded into another BioCell. Each sam-ple was read at 260, 280 and 320 nm on the Epoch spectrophotometer. Backgrounds were corrected using a TE buffer blank at 260 nm, and all concen-tration data was normalized to a 1 cm pathlength and 50 ng/ºL/OD. Sixteen replicate measurements of each standard were used to determine average concentrations, which were then plotted to deter-mine linearity vs. BioCell determinations. As seen in Figure 1, the data is extremely linear, with an R 2 value of 0.998. Additionally, the calcu-lated slope is equal to 1.02, which is indicative of a 2% accuracy difference between micro-volume and BioCell measurements and thus a good equivalence between the data sets. B Comparison of micro-volume 0.5 mm pathlength Take3 versus standard 1 cm pathlength BioCell absorbance measurements on the Epoch spec-trophotometer. A) Full [dsDNA] range from 4-3100 ng/ºL; B) Reduced view of same data to [dsDNA] range from 4-130 ng/ºL. A Figure 2 B Take3 data quality at typical upper and lower limits of quantification Take3 was used to measure dilute (36 ng/ºL) and concentrated (2050 ng/ºL) herring sperm dsDNA standards to demonstrate the wide detection range available with micro-spot measurements. These concentrations are indicative of typical dsDNA con-centration yields from genomic DNA and DNA plasmid isolation kits, respectively. Two microliters of each undiluted concentration were spotted on all sixteen Take3 microspots, and read at 260, 280 and 320 nm on the Epoch spectrophotometer. Backgrounds were again corrected using a TE buffer blank at 260 nm, and all data was normal-ized to a 1 cm pathlength. Figure 2 shows excellent reproducibility from all micro-spot locations for both the dilute and concen-trated samples. An average of 0.075 and 1.8 OD was obtained for the respective samples, and the %CV across the sixteen microspots was 2.3% and 1.8%, respectively. Concentration determinations of A) 36 ng/mL and B) 2050 ng/ºL herring sperm dsDNA samples using all 16 micro-spots of a Take3 plate. Summary Rapid, simple and accurate spectrophotometric nucleic acid determinations are essential in molec-ular biology and other disciplines. Vertical path-length micro-volume absorbance determinations using the Take3 microplate accessory require a min-imum of sample, and eliminate the need for sample dilutions or correction factors, for highly accurate determinations with low error. Bio Research International January-February/2011 10

Micro-Volume Nucleic Acid Determinations with a Microplate Accessory

Peter J. Brescia

Nucleic acid quantification and purity assessment are common, yet critical, methods in molecular biology. Typically, spectrophotometric measurements made at wavelengths of 260 nm and 280 nm provide concentration and purity information - both essential to downstream application requirements for PCR, RT-PCR, qPCR, sequencing, restriction digestions, ligations etc.

Spectrophotometric nucleic acid determinations have traditionally been made in glass, quartz or UV-transparent cuvettes with a fixed, horizontal pathlength of 1 cm. Low volume cuvettes are also available typically with a 1.0 cm pathlength. Spectrophotometric cuvettes typically require volumes of 0.5 to 3.5 mL. The fixed horizontal pathlengths of traditional cuvettes allow single rapid determination of concentration.

Microplate spectrophotometers and UV-transparent microplates now provide increased throughput with the capability for rapid measurement of 96 or 384 samples, with volumes from 10 to 350 ƒÊL. In a microplate, the sample pathlength is vertical, and varies based on the volume used. Pathlength normalization to a 1 cm standard is required for rapid quantification of microplate based measurements to compensate for volume variations, in accordance with the Beer-Lambert Law. It is often necessary to dilute samples for measurement in a microplate or cuvette, as the absorptivity of nucleic acids at 260 nm is high.

A new alternative to cuvette- or microplatebased nucleic acid and protein determinations is a micro-volume device such as the Take3!" Multi- Volume Plate (BioTek Instruments, Inc). The Take3 plate allows up to sixteen 2 ƒÊ L samples to be measured quickly and easily in a microplate spectrophotometer. The Take3 plate also can measure sample in a cuvette or in BioTek s patented 1 cm BioCell!" And is suitable for colorimetric and fluorescent measurements in a microplate reader.

The micro-volume capability of the Take3 plate allows for conservation of precious sample and eliminates the need for dilution. The Take3 microspots have a nominal 0.5 mm fixed vertical pathlength. The Take3 uses two non-boronated glass slides that generally resist non-specific binding. The bottom slide of the Take3 plate is coated with a hydrophobic surface in a 2 x 8 pattern containing 2 mm sample micro-spots . The hydrophobic coating facilitates proper sample droplet formation; when the cover of the plate is closed, the sample droplet is sandwiched between, forming the nominal 0.5 mm pathlength for each sample. After measurement, the sample can be removed and returned to its original vessel, or it may be simple wiped clean.

The short 0.5 mm pathlength provides increased accuracy particularly at upper detection limits compared to that of a 1 cm pathlength, so nucleic acid and protein samples do not require dilution to fall within narrow detection limits. For colorimetric methods, the standard curve and unknown sample data may be generated at the same time, reducing variability from incubation time, temperature and sample manipulation.

Comparing Take3 data to 1 cm pathlength methods

Take3 micro-volume performance was compared to BioCell cuvette performance using an Epoch!" Microplate Spectrophotometer. This comparison serves to confirm that Take3 micro-spot measurements are equivalent to traditional 1 cm pathlength measurement methods.

The unique quartz BioCell allows for absorbance readings with a fixed 1 cm vertical pathlength in BioTek s microplate readers. The monochromatorbased Epoch Microplate Spectrophotometer measures absorbance from 200-999 nm in 6- to 384-well microplates, the Take3 Multi-Volume plate, and the BioCell. Along with endpoint readings for nucleic acid and protein quantification, Epoch can also perform spectral scanning, kinetic and well area scanning measurements.

Concentrated herring sperm double-stranded DNA (dsDNA) in TE buffer (10 mm TRIS, 1 mm EDTA, pH=7.0) was serially diluted to create a series of standards with a concentration range over three orders of magnitude. Two microliters of each undiluted standard concentration was loaded five times at each Take3 micro-spot location using an 8- channel manual pipette. Undiluted standard concentrations or standards diluted 20-fold in TE buffer, were loaded into another BioCell. Each sample was read at 260, 280 and 320 nm on the Epoch spectrophotometer. Backgrounds were corrected using a TE buffer blank at 260 nm, and all concentration data was normalized to a 1 cm pathlength and 50 ng/oL/OD. Sixteen replicate measurements of each standard were used to determine average concentrations, which were then plotted to determine linearity vs. BioCell determinations.

As seen in Figure 1, the data is extremely linear, with an R 2 value of 0.998. Additionally, the calculated slope is equal to 1.02, which is indicative of a 2% accuracy difference between micro-volume and BioCell measurements and thus a good equivalence between the data sets. Take3 data quality at typical upper and lower limits of quantification

Take3 was used to measure dilute (36 ng/oL) and concentrated (2050 ng/oL) herring sperm dsDNA standards to demonstrate the wide detection range available with micro-spot measurements. These concentrations are indicative of typical dsDNA concentration yields from genomic DNA and DNA plasmid isolation kits, respectively. Two microliters of each undiluted concentration were spotted on all sixteen Take3 microspots, and read at 260, 280 and 320 nm on the Epoch spectrophotometer. Backgrounds were again corrected using a TE buffer blank at 260 nm, and all data was normalized to a 1 cm pathlength.

Figure 2 shows excellent reproducibility from all micro-spot locations for both the dilute and concentrated samples. An average of 0.075 and 1.8 OD was obtained for the respective samples, and the %CV across the sixteen microspots was 2.3% and 1. 8%, respectively.

Summary

Rapid, simple and accurate spectrophotometric nucleic acid determinations are essential in molecular biology and other disciplines. Vertical pathlength micro-volume absorbance determinations using the Take3 microplate accessory require a minimum of sample, and eliminate the need for sample dilutions or correction factors, for highly accurate determinations with low error.

Read the full article at http://www.mydigitalpublication.com/article/Micro-Volume+Nucleic+Acid+Determinations+with+a+Microplate+Accessory/655456/62617/article.html.

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