Nucleic acid quantitation and quality control
Methods for quantitation
Uses A260/A230 and A260/A280 ratios. A core operated service using a NanoView (GE Healthcare) spectrophotometer. Provide 2 µL of sample. Request NanoView service under Nucleic Acid -Isolation &Measurement in iLab.
This method is less subject to interference from other materials. This a core operated service using a Qubit Fluorometer (Thermo Fisher Scientific) using a dsDNA HS kit, appropriate for quantitation of PCR products, viral DNA, and NGS samples. Provide 2 µL of sample. Request Qubit under Nucleic Acid -Isolation &Measurement service in iLab.
For large numbers of samples, researchers can perform their own measurement with a fluorescent plate reader (96 or 384 well plates) in the adjacent Biomolecular Analysis Facility. This is a used operated service after training, and requires the researcher to provide reagents and supplies; fee for use.
Methods for size determination, quantity and purity
DNA. The Agilent Bioanalyzer determines size and quantity of nucleic acids by a size based electrophoretic separation and comparison to a known ladder. We currently only stock cartridges for DNA analysis. The DNA 1000, 7500, 12000 cartridges separate DS DNA between 25 and 12,000 base pairs. Sample concentration should be 0.5-50 ng/µL. The DNA HS kit (50-7000 base pairs) needs 5-500 pg/µL. This is a core operated instrument. Please provide 1.5 µL of sample. Fees are charged per sample, but since each cartridge holds 11 (DNA HS) or 12 (all other cartridge types) samples, you may be charged for additional samples if you are not running a full cartridge and we do not have enough other samples to fill the entire cartridge.
To use this service, in iLab, under Request Services, select an Agilent project request.
RNA. The Agilent 4200 TapeStation determines size and quantity of nucleic acids by a size based electrophoretic separation and comparison to a known ladder, much like the Bioanalyzer. We currently only stock the RNA kit for this instrument (RNA of 100-6000 bases). This is a core operated instrument. Please provide 1.5 µL of sample. Users will be sent a PDF via email containing sample results and RIN value at the completion of the run. To use this service, in iLab, under Request Services, select an Agilent project request.. [/ one_third]