DNA Quantitation: Methods and Recommendations in use at GenVault

Correct quantitation of small amounts of DNA is very important for a wide variety of molecular biology applications. Quantitation of DNA is simple in theory but can be difficult in practice. Although every laboratory can quantitate DNA, the process must be effectively standardized and controlled to achieve accurate results. Proper DNA quantitation is the key to good molecular biology results, and failure to produce high-quality data can sometimes be directly attributed to an incorrect estimate of the concentration of DNA template used. It is time-consuming and expensive to apply complicated molecular biology methods to DNA that has not been properly quantitated. Thus, this application note seeks to delineate key advantages, disadvantages and differences in common laboratory quantitation methods including spectrophotometric measurements, fluorometric measurements and agarose gel analysis. The use of quantitative real-time PCR and agarose gel analysis as quality control methods will also be discussed. GenVaults recommendation for use of these various DNA quantitation and quality control methods will be summarized at the end of the article.

Spectrophotometric measurements Background A UV/Vis spectrophotometer measures the intensity of light passing through a sample (I) and compares it to the intensity of light before it passes through the sample (Io). The ratio of I to Io is the transmittance, and is usually expressed as a percentage (%T). The absorbance, A, is based on the transmittance and is derived using the following formula: A = -log(%T) The basic parts of a spectrophotometer are a light source (often an incandescent bulb for the visible wavelengths and a deuterium arc lamp for the ultraviolet range), a sampleholder, a diffraction grating or monochromator to separate the different wavelengths of light, and a detector. The absorbance of UV light as measured in a spectrophotometer can be combined with the following concentration factors to enable calculation of the concentration and purity of dsDNA, ssDNA, and RNA: One A260 unit of double-stranded DNA = 50 g/mL One A260 unit of single-stranded DNA = 33 g/mL One A260 unit of RNA = 40 g/mL Even after DNA is isolated from an organism, protein frequently remains present in the DNA solution. Protein is

tightly bound to DNA and complete removal is not always possible. Both protein and DNA absorb UV light, but have different absorbance curves. The peak of light absorption for DNA is at 260 nm, while protein absorbs at 280 nm, mainly due to tryptophan and tyrosine side chains. When a solution contains both protein and DNA, absorbance at 260 nm is primarily due to the DNA present, but a small amount is due to the protein. At 280 nm, the absorbance is predominantly due to the protein present. Thus, the purity of the DNA can be calculated by examining the ratio of the two absorbance values. A260/A280 values of ~1.7 to 1.8 predict clean DNA, whereas lower values may be indicative of significant protein contamination. However, the A260/280 ratio is not always an accurate representation of DNA purity. Certain samples may be very difficult to evaluate at A280 due to interference from aromatic organic compounds such as phenol, added enzymes, protein stabilizers or crosslinked proteins in samples derived from fixed tissue. Furthermore, some proteins, such as histones and protamines, contain few or no aromatic residues and have little or no absorbance at 280 nm. Therefore, peptide bonds, which absorb at 228 nm, are often a more constant indicator of the presence of protein in a sample. Thus, absorbance readings measured both at 230 nm and at 280 nm provide a more accurate estimate of proteins or peptides that may be present in nucleic acid samples. The ratio of the A260/A230 should be >1.5 since nucleic acids have an absorbance minima at 230 nm1. GenVault recommends taking absorbance readings at A260, 280, and 230 and examining both A260/280 and A230/280 ratios for every sample. Buffer salts can contribute significantly to absorbance readings, especially below 260 nm. Thus, it is imperative to blank the spectrophotometer in the same buffer in which your DNA sample is diluted. Matched glass cuvettes are also important as slight imperfections in the glass may influence readings. Disposable cuvettes should be of high quality and replicate readings should be made to make certain that slight cuvette imperfections are not affecting the absorbance readings. Reliable spectrophotometric DNA quantitation absorbance readings should be in a linear range between 0.1 and 1.5. When working outside these values, quantitation is not valid. Readings greater than 1.5 should be diluted to obtain absorbance values between the acceptable range of 0.1

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and 1.5. Instrumentation and Practical Application A common spectrophotometric instrument used in the laboratory is the NanoDrop. It is available as the NanoDrop 1000, which is a single-measurement (one sample evaluation at a time) full-spectrum UV/Vis spectrophotometer. The NanoDrop 8000 is also a fullspectrum UV/Vis spectrophotometer that can measure eight samples at a time. The NanoDrop 3300 is a fluorometer with a broad excitation range that enables the use of microreactions for quantitation of limited mass3. For the purposes of this article, the NanoDrop 1000 will be discussed because GenVault uses this particular model and finds that many of its customers also use it. With the NanoDrop, the sample is pipetted directly onto the measurement surface. No cuvette is used. Using a patented retention system, a column is drawn between the ends of two optical fibers to establish the measurement path. The technology was developed for use in DuPont Agricultural Genomics Laboratories and has been in full-time use since 1998. Per manufacturer, the Thermo Scientific NanoDrop 1000 spectrophotomer functions as a fullspectrum spectrophotometer covering the range from 220 nm to 750 nm. According to the manufacturer, the 0.2 mm path gives the NanoDrop 1000 the capability to measure DNA concentration up to values 50X greater than that of conventional spectrophotometers, which employ a 1 cm pathlength. However, the very narrow path length of the NanoDrop instrument compromises its ability to measure highly dilute DNA samples. The manufacture-stated dynamic range for the NanoDrop instrument is large: 2 - 3,700 ng/l of dsDNA. In practice, GenVault finds that DNA concentrations of less than 10 ng/l are seldom reliable as the basis for A260-based DNA concentration determination (see Fluorometric measurements section and Table 3 for explanation), and DNA concentrations of less than 20 ng/l are seldom reliable as the basis for the more demanding analysis of DNA purity via A260/A280 or A260/A230 ratios. Table 1 shows an example of NanoDrop absorbance quantitation data from 15 identical genomic DNA samples which were concentrated by YM-100 columns following recovery from GenPlates. Each sample was quantitated in triplicate. Due to variations in filtration speeds of the YM-100 columns, elution volumes for this sample set ranged from 5

61 l, resulting in concentrations ranging from 9.6-124 ng/ ul. In all cases, low A260/280 ratios (<1.6) correlated with DNA concentrations below 20 ng/l. Following NanoDrop quantitation, the indicated samples were subjected to a second YM-100 concentration and eluted in less than 25 l. The NanoDrop quantitation was repeated, resulting in concentrations ranging from 24.2 46.4 ng/l and A260/280 ratios above 1.6 for all samples.
Table 1. Low A260/280 ratios are correlated with DNA concentrations below 20 ng/l.

1st NanoDrop Quantitation NanoDrop NanoDrop Volume Sample Average Average (l) (ng/l) A260/280 1 42 11.3 1.3 2 20 27.7 1.6 3 41 17.5 1.3 4 55 12.1 1.2 5 60 10.0 1.3 6 5 124.0 1.7 7 13 55.2 1.7 8 19 28.0 1.6 9 27 22.3 1.6 10 6 108.1 1.7 11 8 78.6 1.7 12 61 9.6 1.3 13 9 72.2 1.6 14 36 16.6 1.5 15 21 25.7 1.6 2nd NanoDrop Quantitation NanoDrop NanoDrop Volume Sample Average Average (l) (ng/l) A260/280 1 12 36.4 1.8 3 12 36.1 1.8 4 11 44.4 1.8 5 11 46.4 1.8 12 15 24.2 1.9 14 16 26.9 1.9

The NanoDrop protocol states that one l can be used for quantitation. GenVault finds that 2 l is more reliable and reproducible. Other laboratory users recommend 1.5 l. As an added benefit, the sample is also recoverable after quantitation.

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GenVault also finds that replicates read within acceptable correlation of variance between samples or controls, 80120% of the expected value. Fluorometric measurements Background The major disadvantages of the absorbance method of nucleic acid concentration are the large relative contribution of nucleotides and single-stranded nucleic acids to the signal, the interference caused by contaminants commonly found in nucleic acid preparations, the inability to distinguish between DNA and RNA, and the relative insensitivity of the assay (an A260 of 0.1 corresponds to a 5 ng/l dsDNA solution). Hoechst (bisbenzimide) dyes are sensitive fluorescent nucleic acid stains that circumvent many of these problems. Hoechst dyes are somewhat selective for dsDNA, do not show significant fluorescence enhancement in the presence of proteins, and allow the detection and quantitation of DNA concentrations as low as 10 ng/ml. Quantitation of DNA can be easily achieved in a fluorometer with the dye, Hoechst 33258, a bisbenzimide DNA intercalator that excites in the near UV (350 nm) and emits in the blue region (450 nm). Sensitivity of the Hoechst 33258 assay is approximately 10 ng/mL when it is used in conjunction with the TBS-380 Mini-Fluorometer. This dye overcomes some of the limits associated with quantitation of dsDNA by absorbance measurements4. However; the AT content of a DNA sample affects Hoechst 33258-DNA fluorescence. Hence, it is important to use a standard similar to the tested samples. Calf Thymus DNA can often serve as a reference for most plant and animal DNA because it is double-stranded, highly polymerized, and is approximately 58% AT (42% GC). For bacterial DNA, a different standard may be needed because the AT content varies widely depending on the species. Intercalating fluorochromes, such as ethidium bromide or Hoechst 33258, selectively bind to dsDNA. The sensitivity of Hoechst 33258 is ~25 ng of DNA per assay, but preferential association with domains of high A-T content or reduced binding to DNA fragments <500 bp may result in skewed analysis. Accurate evaluation may require sophisticated or dedicated equipment since both ethidium bromide and Hoechst 33258 photobleach easily and fluorescence

enhancement of DNA binding is low, leading to high background readings. Furthermore, these compounds are carcinogenic and pose handling and disposal problems. Fluorescent Dyes and Practical Application Invitrogens Quant-iT PicoGreen dsDNA reagent enables researchers to quantitate as little as 25 pg/ml of dsDNA with a standard spectrofluorometer utilizing fluorescein excitation and emission wavelengths. Per manufacturer, the sensitivity exceeds that achieved with the Hoechst 33258-bases assay by 400-fold5. PicoGreen is a fluorochrome that selectively binds dsDNA and has characteristics similar to that of SYBR-Green I. It has an excitation maximum at 480 nm (lesser peaks in the short-wave UV range) and an emission peak at 520 nm. When bound to dsDNA, fluorescence enhancement of PicoGreen is exceptionally high and little background occurs since the unbound dye has virtually no fluorescence. According to the manufacturer, PicoGreen is somewhat stable to photobleaching. Using the Quanti-it PicoGreen dsDNA reagent and the recommended assay protocol, dsDNA concentration can be analyzed in the presence of equimolar concentrations of ssDNA and RNA with minimal effect on the quantitation results. GenVault uses PicoGreen as the first quantitation choice for the majority of its sample types. Replicates of three are typically run with lower-end standard curves (500 ng/ ml down to1.9 ng/ml) Nine standards are prepared from well H1 to H9 in a standard black 96-well plate suitable for fluorometry. 198 l of TE is added to H1, and 100 l TE is added to the rest of Row H. Wells H10, H11, and H12 serve as blanks or negative controls. Two l of Standard Stock DNA (100 g/ml or 100 ng/l) is then added to H1 and pipette-mixed 10-12 times. The standards are serially diluted 1:1 from H1 to H9 by taking 100 l from H1 and transferring to H2. The well is pipette-mixed 10-12 times, 100 l is transferred from H2 to H3 and pipette-mixed again. This is continued until all samples are diluted through H9. Finally, 100 l is taken out from H9 and discarded. This results in a standard curve from 500 ng/ml serially-diluted by a factor of two down to 1.9 ng/ml (500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125 ng/ml). Positive controls are placed in triplicate on the PicoGreen plate in row G1-G12. Controls used are: 1 ng/l, 10 ng/l, 25 ng/l, and 50 ng/l. For the lower-end assay described

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here, 10 l of the 1 ng/l control is used for analysis, and the 10 ng/l, 25 ng/l, and 50 ng/l controls are diluted 1:10 prior to analyzing 10 l of each sample. Thus, these four controls will cover the low, mid, and high ends of this lowerend standard curve. Yields from DNA extraction of whole blood elements with GenSolve will routinely be in the 1-2 ng/l range in a 200 l final volume. PicoGreen works very well in this range, especially when, as described above, samples are measured in triplicate, with positive controls on the plate in the 1 ng/l to 50 ng/l range. With appropriate dilution, samples with very concentrated values, such as 350 ng/l, can also be read with the PicoGreen quantitation method. Invitrogens Qubit is a relatively inexpensive, portable fluorometer that utilizes the Quant-iT PicoGreen dsDNA reagent with a similar method to the PicoGreen protocol described above. Using the Quant-iT dsDNA HS (high sensitivity) assay kit, the Qubit can detect DNA ranging from 10 pg/l -100 ng/l, according to the manufacturer. Analysis of DNA samples ranging from 0.09 3.11 ng/l revealed that concentrations obtained using the Qubit Fluorometer correlate strongly with concentrations obtained using PicoGreen (Table 2).
Table 2. Comparison of DNA concentration measurements obtained via PicoGreen analysis performed on a Tecan Genios Plate reader or Qubit Fluorometer.

is >10 ng/l. As stated above, spectrometric readings, unlike PicoGreen readings, are not specific for double-stranded DNA. If the PicoGreen and NanoDrop readings do not correlate, the NanoDrop readings are usually higher than PicoGreen, indicating that the DNA sample may contain a mixture of double- and single-stranded DNA, contaminants which scatter light, or UV-absorbing materials that are not nucleic acids. Table 3 shows data from 16 replicate genomic DNA samples with concentrations higher than 20 ng/l, which have highly correlated PicoGreen and NanoDrop values, indicating that the samples contain primarily pure, double-stranded DNA.
Table 3. Comparison of PicoGreen and NanoDrop quantitation measurements in DNA samples greater than 20 ng/l in concentration.
PicoGreen Sample Average (ng/l) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 38.4 37.0 35.6 118.1 102.4 37.5 23.9 35.6 44.1 37.9 33.5 40.1 37.1 46.2 37.2 28.3 NanoDrop Average (ng/l) 39.0 41.2 34.5 103.0 90.0 35.6 23.7 37.2 44.1 38.4 33.4 41.4 39.5 45.9 40.2 29.3 NanoDrop Average A260/280 1.6 1.7 1.6 1.8 1.6 1.7 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.7 1.6 1.6

Sample 1 2 3 4 5 6 7 8 9 10

PicoGreen Average (ng/l) 0.71 0.79 0.93 2.27 0.34 2.31 1.42 1.59 0.35 1.89

Qubit Average (ng/l) 0.69 0.79 0.88 2.21 0.40 2.27 1.34 1.31 0.31 1.63

Correlation Between Fluorometric and Spectrophotometric Methods PicoGreen and NanoDrop values correlate nicely if the DNA is mostly double-stranded and the DNA concentration

PicoGreen is very accurate in the range of DNA concentration below 10 ng/l; however, NanoDrop and other spectrophotometer readings are not accurate in this range. Table 4 shows an example of quantitation of DNA extracted from whole blood spots with low white blood cell counts; and thus, low DNA concentrations. PicoGreen analysis of each sample in triplicate resulted in average concentrations ranging from 0.37 - 4.19 ng/l. However, triplicate readings of the same samples using NanoDrop resulted in average concentrations ranging from 3.77 7.40, with up to a 10-fold inflation of DNA concentration occurring for some samples. Thus, GenVault would accept the PicoGreen data, but not the NanoDrop data in this sample set.

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Table 4. Comparison of PicoGreen and NanoDrop quantitation measurements for DNA samples less than 10 ng/l in concentration.
PicoGreen Average (ng/l) 0.37 1.31 4.19 0.74 1.39 2.47 0.61 2.15 3.17 NanoDrop Average (ng/l) 3.77 5.70 7.83 5.10 5.67 5.97 4.33 6.30 7.40

accumulation of amplicon, an increasing amount of probe hybridizes to target DNA. For the ABI TaqMan probes, a hydrolysis probe, fluoresces, then increases as the 5nuclease activity intrinsic to Taq polymerase hydrolyzes the 5 nucleotide (with fluorophore), freeing it from critical spacing with the quencher associated with intra-molecular energy transfer. Molecular Beacons are hybridization probes. Their fluorescence increases on hybridization to accumulating amplicon, as quenching associated with their stem-loop structure is overcome. Practical Application GenVault does not use qPCR for routine quantitation of DNA, as PicoGreen analysis is less costly and time-consuming. However, GenVault does use qPCR as a quality control measure for detecting contaminants in the DNA sample. GenVault uses the ABI 7700 from Applied Biosystems for qPCR with the TaqMan PCR Reagent Kit with Controls (Applied Biosystems). The reactions amplify a region in exon 3 of the human -actin gene, which is detected by a FAM-dye-labeled -actin TaqMan probe. A VIC-dye-labeled probe/target is used as an internal control to detect the presence of PCR inhibitors. To detect PCR inhibitors, a volume of DNA sample equal to 10% of the reaction volume is typically used, providing a sensitive measure of DNA purity. PCR inhibitors in the DNA sample can be identified by an increase in ct value or amplification failure of the internal control target in samples when compared to the no template control. A known quantity of high-purity DNA is used as a positive control, which provides a comparative ct value for the -actin target. Agarose Gel Analysis Background DNA bands on an agarose gel stained with ethidium bromide or SYBR-Green can be compared to DNA standards to estimate DNA concentration values as described below. However, this method is impractical for routine or high throughput DNA quantitation7. Ethidium bromide, diluted to a final concentration of 0.2 g/ ml or SYBR-Green I, diluted 1:10,000, is used for staining of agarose gels. The gels can be imaged by 254 nm UV transillumination. Commercially-purchased genomic DNA or mass-ladders can be used for calibrating DNA content.

Quantitative Real-Time PCR (qPCR) Background Quantitative real-time PCR (qPCR) allows quantification of starting amounts of DNA, cDNA or RNA templates. qPCR is based on the detection of a fluorescent reporter molecule that increases as PCR product accumulates with each cycle of amplification. Fluorescent reporter molecules include dyes that bind double-stranded DNA (i.e.SYBR Green I) or sequence-specific probes (i.e. Molecular Beacons or TaqMan Probes)6. There are two basic options when choosing a reporter fluorophore: The first, simplest and least expensive is the intercalating dye, SYBR Green I, the fluorescence of which is very low in the absence of double-stranded DNA and very high in its presence. It can be used for any PCR product. A disadvantage is that it will also detect primer-dimers if the PCR reaction is not optimized properly. The second and more sensitive option comprises one of a collection of fluorophore-labeled oligonucleotide probes (e.g., TaqMan, Molecular Beacons or Scorpion probes) designed to anneal near the middle of an accumulating amplicon. Probes contain a reporter fluorophore, e.g., 6-FAM, typically at the 5end, and a quenching fluorophore or blackhole (non-fluorescing) dye at the 3 end. Fluorescence in the absence of amplicon is low, because of intra-molecular quenching optimized in the design of olignonucleotide probes. Labeled probe is in excess of amplicon, at least during the exponential phase of amplification. During

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The standard and control DNAs are loaded on the gel along side the unknown samples. Values for DNA concentration can be obtained visually by comparing fluorescence of DNA bands or obtained quantitatively using densitometry. The amount of sample DNA loaded can be estimated by visual comparison of the band intensity with the standards. More precise agarose gel quantitation can be achieved by densitometric measurement of band intensity and comparison with a standard curve generated using DNA of a known concentration. The intensity of ethidium bromide staining is relatively weak for DNA quantities less than or equal to 10 ng; thus, these small quantities may not give reliable values. In most experiments, the effective range for comparative densitometric quantitation is between 20 and 100 ng2. Per manufacturer, SYBR-Green I is more sensitive than ethidium bromide with a detection limit of ~50 pg per band or ~250 pg per lane of dsDNA. Argon laser-activated gel scanning or capillary electrophoresis is more sensitive, but far more costly7. Practical Application GenVault routinely runs 30 ng (per PicoGreen determination) of unamplified DNA on an 0.8% E-gel (Invitrogen) for 30 minutes to determine DNA length and verify PicoGreen DNA quantitation values. The volume of DNA per well is limited to 25 l so the DNA must be at least 1.2 ng/l in concentration so that the maximum of 25 l can be loaded and DNA visualized. The gel is stained with ethidium bromide. For the purposes of such low resolution analysis, advantage is taken of the roughly logarithmic sieving mechanism of large pore size gels such as 0.8% E-gel or 0.8% agarose. For such gels, DNA smaller than about 35-40 kb is fractionated by size. Above about 35-40 kb, DNA (independent of size) converges to the mobility limit as a single gel band, with apparent mobility equal to about 40 kb, relative to external standards. Figure 1 shows a quality control analysis of ten DNA samples recovered from GenPlates using the GenSolve protocol, which includes a final purification using Qiagen spin columns. Thirty ng of each unamplified genomic DNA sample (per PicoGreen determination) was loaded in lanes 2-10. A ladder with the highest band at 35 kb (APEX) was loaded in lane 1. Thirty ng of human genomic DNA (Roche) was loaded in lane 2 as a positive control. All ten DNA

samples recovered from whole blood stored on GenPlates migrate as single discrete bands in the 35 kb range. The Roche genomic DNA (Lane 2) is cited by the manufacturer as having a length >100kb, whereas Qiagen cites any DNA eluted from its spin columns (lanes 3-12) as having a maximum length in the 35-50kb range, due to limited shearing. Thus, these data confirm that all samples tested migrate as identical gel bands which are 35 kb in size, with little evidence for fragmentation in the samples recovered from GenPlates. Such samples would be described as being full-length and of good quality. Comparing sample band intensities with the Roche control sample band intensity also verifies the PicoGreen quantitation to be correct. When done with some care, this simple 0.8% E-gel (or similar 0.8% agarose) assay can be seen to be a powerful primary screening assay for the integrity of DNA strands, and a useful secondary confirmation of DNA quantitation. This simple gel assay will confirm that a DNA sample is greater than about 35-40 kb in length, which is at least 10 times longer than the strand length required for microarray or most PCR based genetic tests.
Figure 1. Use of agarose gel electrophoresis to confirm DNA concentration and assess DNA length and integrity. Unamplified genomic DNA sample was run for 30 minutes on a 0.8% E-gel. Lane 1: DNA ladder, Lane 2: Roche control DNA (30 ng), Lane 3-12: DNA samples recovered from GenPlates (30 ng).

35 kb

Discussion In various instances, GenVault uses all the four methods discussed above (summarized in Table 5) for quantitation and quality control of DNA samples. PicoGreen is commonly used first for quantitation, and is a very reliable and accurate method for measuring a wide range of DNA concentrations. Samples with a DNA concentration greater than1.2 ng/l

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may be run in a volume of 25 l (30 ng) on a 0.8% E-gel (or 0.8% agarose gel) and compared to known control DNA to verify PicoGreen values specific for dsDNA and to determine maximum DNA length in bp. Full-length DNA stored for years at room temperature (as crude blood or purified DNA) then extracted by GenSolve and purified using Qiagen spin columns is routinely seen to be >35 kb, and is easily visualized on a gel. Samples with DNA concentration values determined by PicoGreen to be greater than 10 ng/l may be quantitated via NanoDrop to verify the proportion of DNA which is double-stranded. If the DNA sample is primarily doublestranded, the PicoGreen and NanoDrop values should match nicely. Samples with DNA concentration values determined by PicoGreen to be greater than 20 ng/l may also be run on the NanoDrop to determine A260/A280 ratios for an estimation of DNA purity. Samples (including those which are ~1 ng/l) may also be analyzed by qPCR to verify that there are no PCR-inhibiting contaminants present in the DNA sample.
Table 5. Summary of quantitation and quality control methods.

the stability of a fluorescent reagent, operator error, poor pipette calibration, etc., as well as obtaining values for DNA quantitation that are in the realm of reality. The goal of correct DNA quantitation is to provide the researcher with proper material for template in downstream applications to further the goal of their research. References
1.http://faculty.ksu.edu.sa/Bazzi-MD/LBCH435/12-DNA%20 Quantitation.doc. 2.QIAGEN News, Issue 2, 1998, Philippe Sauer, Markus, Mller, and Jie Kang. 3.http://www.nanodrop.com/. 4.http://www.turnerbiosystems.com/doc/appnotes/s_0072.htm. 5.Invitrogen Product information sheet for Quant-iT PicoGreen dsDNA Reagents and Kits (Molecular Probes). 6.http://www.sigmaaldrich.com/Life_Science/Molecular_Biology/ PCR/Product_Lines/Quantitative_PCR.html. 7.Ahn SJ, Costa J, and Rettig Emanuel J. PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR. Nucleic Acids Res. 1996 24(13) 2623-2625.

Method
UV/Vis Spectrophotometry with NanoDrop Fluorometric Measurement with PicoGreen Quantitative RealTime PCR Agarose Gel Analysis

Minimum Concentration
10 ng/l 20 ng/l 25 pg/ml

Primary Application
Concentration Purity

Concentration
Detection of PCR inhibition Fragment length

1 ng/l 1.2 ng/l (30 ng)

Conclusion As noted above, all four types of DNA quantitation are used by GenVault for accurate DNA quantitation and quality control. It is imperative that appropriate standards and controls are run with each assay, generally in triplicate. Standards alone are not a substitute for controls. No quantitative DNA data at GenVault is reported without appropriate controls and standard correlation. Close attention to details in standard curves and control values will assist in identifying trends such as a failing light source in a spectrophotometer, erratic blank readings, changes in

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