Sample Drop-off Hours: M-F 7:00am-5:00pm
GHTF provides several services for checking the quality of DNA and RNA samples. Samples can be quantified via: Qubit assay, nanodrop, or qPCR. The quality of DNA and RNA samples can be checked with Aglient’s Bioanalyzer.
Nanodropping is a common way to quantify DNA or RNA using UV absorbance. UV light is shown through 2ul of sample, and the spectrophotometer then records the light absorbance at different wavelengths. Nucleic acids absorbs at 260nm and proteins absorb at 280nm. By looking a the 260/230 and 260/280 ratios, it is also possible to approximate the amount of contamination in the sample.
Qubit quantification works by using fluorescence dyes that specifically bind to DNA or RNA. Ideally, the fluorescence detect should only be coming from the target of the assay, so quantification by qubit are typically more accurate than quantification by UV absorbance. Types of Qubit assays offered at GHTF are dsDNA High Sensitivity (0.2-100ng), dsDNA Broad (2-1000ng), and RNA High Sensitivity (5-100ng). Please note that as the concentrations get lower, more sample volume (up to 20ul) is required to be within the qubit quantifiable range.
Kapa qPCR Quantification
Kapa qPCR can be used to quantify Illumina compatible libraries. An aliqout of the library undergoes PCR amplification with the addition of fluorescent dyes. This allows the machine to determine the Ct value or cycle quantification value, which is the PCR cycle number at which the machine detected a real signal from the library. This method makes Kapa quantification a rather accurate means to quantify Illumina libraries because only the DNA with Illumina Universal adapters ligated will get measured.
The bioanalyzer like a small scale gel electrophoresis, using a micro-capillary electrophoretic chip. The machine runs the samples and plots the fluorescence detected at each size (nt or bp). The kits available at GHTF are: RNA Pico, RNA Nano, DNA High Sensitivity, and DNA 12000. The difference between RNA Pico and Nano is the quantification range: Pico (0.1-5ng/ul) and Nano (25-500ng/ul). The difference between DNA High Sensitivity and DNA 12000 is also sensitivity, however there is also a difference in the range of DNA sizes: DNA High Sensitivity (0.5-3ng/ul and 50-7000bp) and DNA 12000 (5-50ng/ul and 100-12000bp).