Services
– Qubit
(DNA, RNA, Protein)
• Instruments
• Self-Service?
• Available Assays
• Service Requirements
• Online Submission Request
• Physical Submission of Samples
With a reservation in Biosci-Booked (Genomics), you may use our Qubit 4.0.jpg with your own Qubit kits; otherwise, make an online submission for processing by the Genomics Core. All Qubit assays require 1-20 ul of sample in Axygen 0.5-ml tubes (PCR-05-C, thin-wall, clear).
Instruments
Qubit 4.0: fluorometer for measuring DNA, RNA, & protein quantity. In addition, with the Qubit RNA IQ assay, the Qubit 4.0 accurately distinguishes intact from degraded RNA by using two unique dyes: one that binds to large, intact RNA, and another that selectively binds to small, degraded RNA. The Qubit 4.0 is in the main room of A628.
Qubit 3.0: fluorometer for measuring DNA, RNA, & protein quantity. This instrument is located in A628-A, and it is normally reserved for use by Core personnel.
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Self-Service?
If using your own kits with our Qubit instruments, reservations through Biosci-Booked (Genomics) are required; in addition, if you wish to use the Qubit 3.0, you must request permission in person.
Otherwise, samples are processed by the Genomics Core; submit samples in accordance with the requirments listed below.
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Available Assays
There are many
Qubit assays available from ThermoFisher. Assays require 1-20 ul of sample, resulting in the quantitation ranges shown with each kit. Samples are analyzed using Axygen 0.5-ml tubes (PCR-05-C, thin-wall, clear).
Create Your Own assay
RNA IQ assay (integrity & quality)
dsDNA HS Assay Kit (0.2-100 ng)
dsDNA BR Assay Kit (2-1000 ng)
ssDNA Assay Kit (1-200 ng)
RNA HS Assay Kit (5-100 ng)
RNA BR Assay Kit (20–1,000 ng)
RNA XR Assay Kit (0.02-8 µg)
microRNA Assay Kit (1-1000 ng)
Protein Assay Kit (0.25-5 µg)
We keep the dsDNA HS Assay kit in stock at all times; see description below. For the other Qubit assays, please inquire to determine if they are currently in stock and if there are any kit-specific requirments.
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Create Your Own assay
The MyQubit functionality allows you to create new assays for the Qubit 4 Fluorometer in minutes, using parameters that can easily be uploaded to the instrument using a USB drive, without changing the existing assays. New assays can be optimized versions of existing fluorescence-based assays or can be completely novel. For details, see
MyQubit Custom Assays on the Qubit 3.0 and 4 Fluorometers.
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RNA IQ assay kit
In combination with the Qubit 4.0, this assay distinguishes intact from degraded RNA by using two unique dyes: one binding to large, intact RNA, and another binding to small, degraded RNA. Results are provided as an
RNA IQ#, similar in concept to the
RIN metric provided by a Bioanalyzer RNA assay; however, for the same sample, the Bioanalyzer's RIN value will tend to be slightly lower than the Qubit's RNA IQ value (reflecting the Qubit assay's inability to distinguish slightly degraded large RNA from completely intact large RNA).
(
Note: This kit is not compatible with earlier versions of the Qubit.)
The Qubit RNA IQ assay also does not provide an estimate of RNA concentration, unlike the Bioanalyzer assay. On the other hand, the Qubit RNA IQ assay is considerably cheaper than running a Bioanalyzer chip, and results can be obtained much faster due to a less demanding setup process for the Qubit assay.
For good accuracy, the RNA IQ assay requires that the assay tube contain 500-1500 ng of RNA; see listing below for the appropriate sample volumes based on your RNA concentrations. To avoid "Measurement Error" readings with this assay, please first use the Nanodrop (or an Qubit RNA quantitation assay) to determine RNA sample concentrations.
- 500-1500 ng/µl ..... use 1.0 µl.
- 250-750 ng/µl ....... use 2.0 µl.
- 100-300 ng/µl ....... use 5.0 µl.
- 50-150 ng/µl ....... use 10.0 µl.
- 33-100 ng/µl ....... use 15.0 µl.
- 25-75 ng/µl .......... use 20.0 µl.
If diluting your samples, note that measurement error is higher when smaller volumes are involved... plan accordingly. Finally, when submitted to the Genomics Core for processing, all tubes must contain the same total volume (RNA, plus any additional buffer) so that a single Master-mix can be prepared for all samples.
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dsDNA HS (High Sensitivity) Assay Kit
This assay is highly selective for
dsDNA over RNA and ssDNA, accurately quantitating initial sample concentrations from 10 pg⁄µl (using 20 µl sample) to 100 ng⁄µl (using 1 µl sample). The kit provides concentrated assay reagent,
dilution buffer, and pre-diluted DNA standards.
Assay Range: Total DNA/sample must be between 0.2-100 ng; samples which fall outside this range will register as either (<0.50 ng/µl) or (>500 ng/µl).
Total volume: Sample must be within 1-20 µl.
Serial Dilutions: Common contaminants, such as salts, free nucleotides, solvents, detergents, or protein are well tolerated in the assay; however, we have found that DNA extractions with Trizol affect the assay. In such cases, we recommend processing a few samples using a serial dilution of those samples to find the optimum volume of sample for generating accurate DNA quantitation.
DNA Standard #2: For both dsDNA assays (HS and BR), the molecule in the Standard #2 vial is Lambda DNA from an E. coli lysogen (48-kb, dsDNA). In the event that you either run out of the official Standard #2 or you wish to test an external 'control' dsDNA (virtually identical to the kit's standard), order Lambda DNA (cIind 1 ts857 Sam 7) — ThermoFisher Product Number 25250010.
DNA Types: The Qubit dsDNA kit is ideal for samples that contain relatively short DNA fragments (e.g., sheared genomic DNA or PCR products). However, for some types of DNA (e.g., plasmids or intact DNA from organisms with very large genomes), results may be erratic or simply offset from the actual DNA concentration. This is probably due to differing levels of supercoiling of the DNA, leading to differential intercalation of the reporter dye into the sample DNA versus the kit's Standard #2. For genomic DNA, it can be helpful to warm the sample (70oC, 2 min) and then cool it to room temperature (5 min) before adding the reagent mix. Because plasmids can shift rapidly among different levels of coiling, concentration estimates can shift dramatically even among repeated readings of the same assay tube; to improve results, it can be helpful to first linearize plasmids with double or triple restriction digests.
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Service Requirements
Tubes: Samples must be submitted in Axygen 0.5-ml tubes (PCR-05-C, thin-wall, clear); packs of 100 tubes are available as a "Supply" on the Request Form for the Genomics Core website.
Sample Volume: you must add the desired volume of sample to each tube and state the volume(s) in the "Client Memo" of your online submission. Ideally, all samples will use the same volume; however, if multiple volumes are used, they must be clearly specified in some way (e.g., in "Client Memo", a note attached to samples, on the tubes, or by email).
Sample Names: Tubes should be numbered on the top; however, if you wish, you may include a list of corresponding sample names. Short lists can be put in the 'Client Memo'; for large submissions, please email the names as an Excel file.
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Online Submission Request
Log In to the website, and click on "Services Page":
- Make the necessary 'selections'.
- In the "Client Memo" box, specify the volume of sample provided and any other relevant information (e.g., short list of sample names; DNA type [see above]; or special instructions).
- Click the 'check box', and submit your request.
- Finally, if desired, email an Excel file of your sample names (corresponding to the numbers on the tops of the tubes).
If you make an error in your request, you may revise that request if you do so before we begin processing it.
You will receive an automated email when the data for your samples have been uploaded to your 'Submission History'.
Files will remain in your 'Submission History' for ≥90 days; if you need a copy of the data after it's purged, we can usually retrieve it from our archives.
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Physical Submission of Samples
Label Submission: Upon successful submission of your online request, the 'Submission #" will appear under your "Submission History". Then, label the tube rack itself with:
- Submission #;
- Date;
- PI's Last Name; and,
- Submitter name or initials.
Delivery site: Transfer tubes to a Genomics Core rack (taking your own rack back to
your lab), and put samples in the Mini-fridge (by sink) in the Genomics Core (A628 Life Science Annex).
[Note: If the Core is locked, put your samples in the plastic "After-Hours" GC drop-box located in the adjacent Cold Room (beside sink, Rm. A650).]
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