1. The world leader in serving science NanoDrop Product Training NanoDrop 1000 Software

2. 2  Computer Requirements  Nucleic Acid Module  A280 Module  MicroArray Module  Proteins and Labels Module  Colormetric Assays  Microbial Cell Culture  General UV/Vis  Data Viewer  Additional modules Topics

3. 3 Microsoft XP or 2000 Operating System. Windows Vista has also been tested successfully with NanoDrop software. The operating software is not compatible with Windows NT, 95, 98 or ME. Minimal requirements, no need for dedicated PC NanoDrop 1000 and NanoDrop 3300 NanoDrop 8000 233 MHz or higher processor 800 MHz or higher processor CD ROM drive 32 MB or more of RAM 128 MB or more of RAM 40 MB of free hard disk space 100 MB of free hard disk space Open USB port (the instrument can only be connected via the USB port) Computer Requirements

4. 4  Nucleic acid concentration and purity (2.0ng/ul-3700ng/ul dsDNA)  Fluorescently labeled oligos for microarray  Protein concentration (A280) (0.1 mg/ml-100 mg/ml-BSA)  Fluorescently labeled proteins and metalloproteins  Colorimetric protein assay (i.e. Bradford, BCA, Lowry)  Microbial cell density measurements  General UV-Vis spectrophotometry Software Modules

5. 5  Nucleic acid quantitation and purity  MicroArray probe preparation  Quantitative RT-PCR  Sequencing  Genotyping  Histocompatibility  Microgenomics Used for Quality Control during Sample Preparation NanoDrop 1000 and NanoDrop 8000 Spectrophotometers Nucleic Acid Module

6. 6  Blank: Reference spectrum  Re-blank: New reference spectrum as well as display of last sample  Measure: Used to measure samples  Recording: Saves data to current report  Sample type: Color coded  Sample ID: Enter prior to sample measurement. Changes through Data Viewer.  260/280 ratio: Sample purity indicator  260/230 ratio: Sample purity indicator  : User selectable wavelength  10 mm path: Normalized NanoDrop 1000 interface

7. 7  Reverse transcriptase-polymerase chain reaction (RT-PCR) amplifies cDNA following its transcription from RNA and can be used when comparing  Different cell lines or tissues  Time course of drug treatment compared to the untreated control  Diseased versus nondiseased tissues  Critical that each reverse transcription reaction in the study contains equivalent amounts of RNA.  Laser capture microdissection (LCM) enables the isolation of desired pure cell populations as limited as single cells from heterogeneous tissue samples.  Preserves essential cellular and morphological characteristics including the integrity of biomolecules such as DNA, RNA, and proteins.  Often very low nucleic acid yield.  The time-limited nature of organ procurement and Human Leukocyte Antigens (HLA) Typing requires instruments that are efficient as well as reliable  Bone marrow transplantation labs can have difficulty acquiring enough mononuclear cells to get good DNA yields.  Dramatic acceptance of Nanodrop ND-1000 in HLA labs in 2 years. Nucleic Acid Applications

8. 8  The A280 Module does not require generation of a standard curve  Six sample type options  10 mm normalized path The A280 method is applicable to purified proteins exhibiting absorbance at 280nm. A280 Module

9. 9 A280 Sample Type Options

10. 10 The capability to pre-select viable fluorescent-tagged hybridization probes for gene expression in MicroArrays can eliminate potentially flawed samples and improve research effectiveness.  Measures the concentration of nucleic acid and the absorbance of up to 2 fluorescent dyes.  Dye number selected using User Preferences  Detects dye concentrations as low as 0.2 picomole per microliter.  1 mm path MicroArray Module

11. 11 Fluorescent labels are useful to biomedical researchers running microarrays, protein arrays, and flow cytometry.  Cy dyes are commonly used long-wavelength dyes (Amersham)  Alexa Fluor dyes are generally more stable, brighter, and less pH-sensitive than common dyes (e.g. fluorescein, rhodamine) of comparable excitation and emission. (Invitogen)  MicroArrays  Composed of a collection of unique DNA probes arranged on a solid substrate.  Probes composed of DNA sequences complementary to the sequence of interest.  Nucleic acid “targets” incorporating fluorescent dyes anneal to the complementary probes.  Differential color or signal intensity correlates with target abundance. Fluorescent Dyes

12. 12   3: User selectable  Normalized to 10 mm path  Also used to measure the purity of metalloproteins (such as hemoglobin) using wavelength ratios. This software module can be used to determine protein concentration (A280nm) as well as up to 2 fluorescent dye concentrations Proteins & Labels Module

13. 13 Assay Protein Concentration Range Advantages of Method Disadvantages of Method Pierce BCA (Bicinchoninic Acid) 20:1 reagent/sample volume 1:1 reagent/sample volume 0.2 to 8.0 mg/ml BSA 10 – 200 ug/ml BSA Compatible with most surfactants Copper chelators, reducing agents may interfere with the BCA assay. Bradford (Coomassie) 50:1 reagent/sample volume 1:1 reagent/sample volume 0.1 to 8.0 mg/ml BSA 15 – 100 ug/ml BSA Fastest and easiest protein assay. Room temperature. Linear range is 0.1-1 mg/ml Surfactants may cause the reagent to precipitate. Twice as much protein-to-protein variation as BCA assay. “Un-Conditions” pedestals. Modified Lowry (Cupric sulfate-tartrate) 0.2 – 4.0 mg/ml BSA Can be measured at any wavelength between 650 nm and 750 nm with little loss of color intensity. Detergents, potassium ions form precipitates. Chelating agents, reducing agents, and free thiols interfere with this assay. Colormetric Assay Modules The Pierce BCA Assay is used for more dilute protein solutions and/or in the presence of components that also have significant UV (280 nm) absorbance. The Bradford Assay response varies with the composition of the protein. The assay is also sensitive to non protein sources, particularly detergents, and becomes nonlinear with higher protein concentrations. The Modified Lowry Protein Assay Folin-Ciocalteu reagent is effectively reduced in proportion to the chelated copper-complexes.

14. 14 Colormetric Modules  Tab structure to view samples or standards  Valid only indicates minimum number of measurements made  Additional cursor position available to measure optional wavelength

15. 15 Step 1: Measure the ‘Reference’ (Reagent only – a ‘zero’ Standard) Step 2: Measure Standards Up to 5 replicates each of up to 7 standards can be measured. Step 3: Measure Samples Sample concentrations can be calculated by using linear interpolation (point-to-point) between the two standards flanking the unknown sample or by using a polynomial fit. Colormetric Assay Standard Curves

16. 16  Curve Fit Options  Interpolation  Linear  2 nd or 3 rd Polynomial  Save and Recall  Store and reuse standard curve  NanoDrop 8000 allows for recall of dilution concentration series NanoDrop Software offers Flexibility when using Standard Curves. Colormetric Assay Standard Curves

17. 17 Bradford Standard Curve Pierce BCA Standard Curve Bradford vs BCA Results BCA is preferred when possible as better dynamic and linear range.

18. 18 Due to its shorter pathlength, the NanoDrop 1000 can measure cell densities that are 10-fold higher than those measurable on a standard cuvette spectrophotometer.  Diluted samples with low ‘Absorbance’ at 600 nm can be monitored at lower wavelengths  (i.e. 320 nm)  Use 2 ul samples  Mix the culture well  Avoid bubbles  Measure quickly to avoid settling Microbial Cell Cultures

19. 19 General UV- VIS  Displays absorbance measurements from 220 nm to 750 nm.  Has 2 cursors to permit measurement of individual peaks  User selectable baseline  User selectable normalization- lowest value 400-750 nm  Hi Abs feature- normalized to 0.1 nm on screen

20. 20  Data Viewer  Account Management  User Preferences  Dye/Chromophore Editor  Utilities and Diagnostics Additional Main Menu Options

21. 21 Data Viewer Data Viewer is a versatile, integrated data reporting software program  Offers the user the ability to customize report structures, import archived data and re- plot data generated from NanoDrop instruments.  All data automatically archived on hard drive.  Accessed from either the Main Menu or the Show Report function

22. 22 Customize Report Structures Re-plot Data Import Archived Data Data Viewer

23. 23 Utilities and Diagnostics Intensity Check Calibration Check Selections

24. 24 Additional Main Menu Options User Preferences Account Management Dye Chromophore/Editor

25. 25 Q: What sort of accuracy and reproducibility, should I expect with the NanoDrop 1000? A: Accuracy, typically within 2%. Reproducibility, typically +/- 0.003 A at low concentrations Q: Is simply wiping the pedestal surface enough to prevent carryover? A: Yes. The highly polished quartz and stainless steel surfaces of the sample retention system are resistant to sample adherence, making the use of dry laboratory wipes very effective in removing the sample. Q: Do nucleic acids require purification prior to measurement on the NanoDrop 1000? A: Yes. Absorbance measurements are not specific for a particular nucleic acid. Q: Are there solvent restrictions? A: Hydrofluoric acid can etch the quartz optical fiber. Most other laboratory solvents typically used in life science labs, including dilute acids, are compatible as long as they are immediately wiped away. Q: How do I check the accuracy of the NanoDrop 1000? A: CF-1 calibration check fluid should be used with our Calibration Check module or software. Q: How often do I need to check the accuracy of the NanoDrop 1000? A: We recommend confirming that the instrument is within calibration specifications every 6 months using the CF-1 Calibration Check Fluid. Q: How long before I need to replace the flashlamp? A: The lamp is rated to last for a minimum of 30,000 measurements before replacement could be required. NanoDrop 1000 FAQs

26. 26  Use a 1.5-2 ul sample size Erroneous results can occur when the liquid sample column is not completely formed during a measurement. Note: Concentration calculations are volume independent.  Ensure sample solution is homogeneous and purified Important to ensure that the sample especially genomic DNA being measured is homogeneous.  Confirm that your sample is within linear range of instrument Measuring samples at or near the detection limit will result in higher CVs.  Confirm that the reference (blank) solution and sample solvent are the same Buffers often absorb in the UV range. Highly volatile solvents may not be conducive for use due to the rapid evaporation and concentration of sample.  Use fresh aliquots for each measurement Multiple measurements of the same aliquot will result in evaporation. Sample Reproducibility