Nucleic Acid Quality/Concentration
The Genome Core has two pieces of equipment to measure nucleic acid quality and concentration: the NanoDrop 1000 spectrophotometer and Agilent 2100 Bioanalyzer.
Nanodrop
Description
The NanoDrop ND-1000 UV-Vis Spectrophotometer enables highly accurate analyses of extremely small samples (1-2ul standard). The system does not require cuvettes or capillaries, which decreases the measurement cycle time. In addition, the high absorbance capability (50 times most spectrophotometers) eliminates the need for most dilutions. It has a large dynamic range (measures 2-3700 ng/ul (dsDNA) on a single sample), and it measures at the full spectrum (220-750nm). (More info from NanoDrop.)
Science
What the 260/280 ratio measures:
- 260nm - absorption of nucleic acids (DNA & RNA)
- 280nm - absorption of proteins (specifically aromatic amino acids)
- 230nm - absorption of organic contaminants and solvents (phenol, thiocyanates, urea, carbohydrates, B-mercaptoethanol, carryover guanidinium from chaotropic lysis buffer)
For RNA:
-
260/280 of pure samples is 2.0 +/- 0.1. As this ratio decreases, protein contamination rises (purity decreases).
- 260/230 of pure samples is between 2.0 and 2.4. Samples with a 260/230 below 1.8 have significant contamination that may affect downstream results such as expression analysis.
For DNA:
-
260/280 of pure samples is 1.8 +/- 0.1. As this ratio decreases, protein contamination rises (purity decreases).
- 260/230 of pure samples is between 2.0 and 2.4.
* Absorbencies and ratios depend on the pH of the sample. Blanks on the Nanodrop should be done with the solvent (i.e. TE, not H20).
Many researchers encounter a consistent 260/280 ratio change when switching from a standard cuvette spectrophotometer to the NanoDrop ND-1000 spectrophotometer. The main causes for this are listed here.
Protocol
Select "ND-1000 User's Manual" from the NanoDrop website.
Pricing
See pricing schedule here.
All users with SOM (School of Medicine) email accounts have instant access to the Nanodrop 1000 and attached computer. To access the software log on to the SOM server with your email and password. A link to a temporary storage folder is available on the server. Do not store data locally, as it impedes the functioning of the Nanodrop software. If you do not have a SOM account, please request a local account one day before you want to use the machine. Send an email with your first name, last name, PI, and department to genome@cc.ucsf.edu. The Core will email you a login and password. Conversely, the Core can measure the concentration of samples for a fee based on an hourly charge.
What to Provide
Please provide the core with the RNA samples or gDNA samples for which you would like the concentration determined. Note that for each sample a MINIMUM of 2ul will be used by the Nanodrop, so for samples which require further analysis (i.e. RT and/or relative expression), please plan accordingly by providing us with extra volume. You will also need to upload the corresponding form on CoreAdmin.
Bioanalyzer
Description
The Agilent 2100 Bioanalyzer uses microfluidic lab-on-a-chip technology to provide sensitive quality analysis of DNA, RNA, proteins, and cells. Digital data are available in easy-to-interpret format. New software provides the RNA Integrity Number (RIN), a quantitative measurement of RNA quality, of which a high value is often required before running samples on microarrays. Degraded RNA can influence the variation in QPCR measurements. For improved sensitivity or troubleshooting due to failures, we recommend the use of the Bioanalyzer to assess RNA quality.
Science
See information from Agilent Technologies.
Protocol
See information from Agilent Technologies.
Pricing
See pricing schedule here.
The Core buys Nanochips (RNA concentration of 1-500 ng/ul) in bulk and provides the reagents and chips at cost to users. We can also run the samples full service for a slightly increased fee. Packages of chips for lower concentrations of RNA (<5ng/ul), DNA, protein, and cells must be purchased by the user.
What to Provide
Please provide the core with the RNA samples you would like run on the Bioanalyzer. A MINIMUM of 3ul (1-500 ng/ul) is required for each sample. Please provide in 1.5ml or 0.5ml eppendorf tubes labeled with sample numbers in pen (NOT TAPE, and NO strip tubes). You will also need to upload the corresponding form on CoreAdmin.
