Nucleic Acid Quantification Support—Getting Started

The NanoDrop® instrument uses UV absorbance, which cannot distinguish between DNA, RNA, free nucleotides, and other contaminants. The Qubit® Fluorometer utilizes specifically designed fluorometric technology using Molecular Probes® dyes to quantitate biomolecules of interest. These fluorescent dyes emit signals ONLY when bound to specific target molecules, even at low concentrations.

UV absorbance readings measure anything that absorbs at 260 nm, including DNA, RNA, protein, free nucleotides, and excess salt. Quantification with the Qubit® Fluorometer only measures the molecule you are interested in, so the number is almost always lower than the A260 reading.

No. The Qubit fluorometer cannot take absorbance readings to provide a A260/A280 ratio or detect protein in nucleic acid samples. This can be done with the NanoDrop instrument. If your sample contains protein or other contaminants that can affect the assay, it should be further purified.

The Qubit DNA and RNA kits only quantify the amount of either DNA or RNA in the sample. If your sample contains both DNA and RNA, one may use either (or both) the DNA and RNA Qubit kits and compare with samples treated with either RNase or DNase to get an accurate determination of DNA or RNA, respectively.

The Qubit® 2.0 Fluorometer has a large, robust touch screen for seamless workflow navigation. It also offers automatic data logging and a USB port for efficient data management.

Yes, these kits will work with all Qubit® Fluorometers. 

The Qubit® 3.0 Fluorometer can store up to 1,000 samples’ worth of data in a .csv file.
The Qubit® 2.0 Fluorometer can store up to 200 lines of data in a .csv file.
The original Qubit® (1.0) Fluorometer can store up to 20 lines of data in a .csv file.

No. But we do recommend using new standards every time you make a new working solution, so that the working solution used in your standards is the same as that used in your samples.

The diluted standards can be used for up to 3 hours if using the same working solution for the samples.

Generally, the cleaner the sample the better. Some salts, proteins, and detergents are tolerated in the assays; see the specific assay protocol for which ones and at what concentrations.

Yes, you can, for Qubit® instruments developed after the original Qubit® (1.0) Fluorometer. See MyQubit assay instructions here.

There are two light sources in the Qubit® 2.0 and 3.0 Fluorometesr - both are LEDs. They are expected to last at least 5 years.

No. The warranty will be voided if the instrument is disassembled or if you attempt to repair the instrument. It is best to call or email our technical support team for assistance.

We will replace the instrument. Please contact our Technical Support department for details by emailing techsupport@thermofisher.com.

For Qubit 4 and older instruments, use thin-walled, clear 0.5 mL PCR tubes such as Invitrogen Qubit Assay Tubes. For Qubit Flex Fluorometers, use thin-walled 200 µL polypropylene tubes such as Invitrogen Qubit Flex Assay Tube Strips. Other types of tubes can have auto-fluorescence or a frosted area for writing and may interfere with the assay.

The USB that comes with the Qubit® 2.0 Fluorometer is 2 GB, and the one that comes with the Qubit® 3.0 Fluorometer is 4 GB, but the following types are also compatible:

• ADISK USB 1.1 32MB flash disk
  
• Aigo USB 1.1 64M flash disk
  
• Crucial 1G flash disk
  
• FPT-D US5B2H01 18-in-1 USB card reader/writer
  
• HP 1G flash disk
  
• IBM Portable Diskette Drive (floppy drive)
  
• Integral USB 2.0 2GB flash disk
  
• Kingston DataTraveler 1GB flash disk
  
• Kingston DataTraveler 100 2GB flash disk
  
• Kingston DataTraveler 16GB flash disk
  
• LACIE USB 2.0 40GB mobile hard drive
  
• Lexar Media JumpDrive Secure USB 2.0 512MB flash disk
  
• Memorex 2GB flash disk
  
• NCP XDrivePlus MMC/SD reader
  
• Newman USB 1.1 64MB flash disk
  
• PNY Attache USB 1.1 64MB flash disk
  
• PNY Attache (U3) 1GB
  
• PNY Attache 2GB
  
• PNY Attache 8GB
  
• PQI MMC/SD reader
  
• RedLeaf USB 2.0 256MB flash disk
  
• SanDisk Cruzer USB 2.0 256MB flash disk
  
• SanDisk Cruzer Micro 2GB flash disk
  
• SanDisk Cruzer Micro (U3) 2GB flash disk
  
• SanDisk Cruzer Micro (U3) 4GB flash disk
  
• SONY MICROVAULT USM256U2 USB 2.0 256MB flash disk
  
• Transcend JF V30 4GB flash disk

The following have not been verified by our R&D staff, but have been tested by customers:

• Edge DiskGO™ 1GB USB Flash Drive Enhanced for ReadyBoost™
  
• Edge DiskGO™ 2GB USB Flash Drive Enhanced for ReadyBoost™
  
• Imation 1GB Swivel USB Flash Drive
  
• Imation 2GB Swivel USB Flash Drive
  
• Integral 1GB USB Memory Stick
  
• MARKEM 1GB USB Memory Stick
  
• Memorex 1GB TravelDrive™ USB Flash Drive
  
• Memorex 2GB TravelDrive™ USB Flash Drive
  
• PNY 1GB Attache USB Flash Drive
  
• SanDisk 2GB Cruzer® Crossfire USB Flash Drive
  
• SanDisk 512MB Cruzer® Micro USB Flash Drive
  
• SanDisk 2GB Cruzer® Micro USB Flash Drive
  
• SanDisk 4GB Cruzer® Micro USB Flash Drive (U3 function not initialized)
  
• Sony 512MB Micro Vault Tiny USB Flash Drive
  
• Sony 2GB Micro Vault Tiny USB Flash Drive
  
• Sony 1GB Micro Vault Classic USB Flash Drive
  
• Sony 4GB Micro Vault Classic USB Flash Drive
  
• X Digital Media 1GB Itty Bit USB Flash Drive
  
• X Digital Media 1GB Poker Chip USB Flash Drive
  
• X Digital Media 2GB Itty Bit USB Flash Drive

• Qubit® dsDNA HS Assay: 502 nm/532 nm
• Qubit® dsDNA BR Assay: 510 nm/527 nm
• Qubit® ssDNA Assay: 493 nm/518 nm
• Qubit® RNA HS Assay: 644 nm/673 nm
• Qubit® RNA BR Assay: 644 nm/673 nm
• Qubit® microRNA Assay: 498 nm/518 nm
• Qubit® Protein Assay: 470 nm/570 nm

The Qubit® 3.0 Fluorometer employs a large, robust color touch screen for seamless workflow navigation and exports data to a USB drive or directly to your computer via a USB cable for efficient data management. Also, the instrument can be personalized to show only the frequently used assays, to add new assays, including user-defined assays created with the MyQubit assay design tool, and to display in the language of your choice including English, French, Spanish, Italian, German, simplified Chinese, and Japanese.

Yes, we expect to deplete the current inventory and discontinue the Qubit® 2.0 Fluorometer in 2015.

Yes, the Qubit®3.0 Fluorometer comes with both a USB and cable to connect to a computer.

The Quant-iT™ PicoGreen® DNA assay is the oldest assay and the most general-purpose. It requires the most user preparation and calculation, but can be adapted for cuvettes or plates. The Quant-iT™ DNA Assays are designed for high throughput (>20 samples) and for use in 96-well plate readers with no further adaptation. The Qubit® assays are intended for low throughput (<20 samples), and are only used on the Qubit® Fluorometer. The Qubit® Fluorometer contains highly optimized algorithms that calculate the concentration of your sample for you. The performance of all of these assays is similar.

Yes, we would recommend reducing the volume while keeping the dye: sample ratio constant.

Yes, short fragments may not stain well and plasmid DNA results will vary depending on conformation (see the following questions). Below is data for the PicoGreen® assay:

Effect of DNA structure on the PicoGreen® assay. The signals obtained using linear calf thymus DNA (closed stars), bacteriophage lambda DNA (closed squares), restriction-digested bacteriophage phi X174 RF DNA (open diamonds), and a commercially available set of size marker DNAs (open circles) were compared with signals obtained using supercoiled pUC19 (2,686 base pairs) DNA (open triangles) and phi X174 RF (5,836 bases) DNA (open stars).

Yes. Use the Qubit® DNA BR Assay for a typical plasmid miniprep with a high concentration of DNA (over 50 ng/μL). Use the Qubit® DNA HS Assay for plasmid rescue or methods that yield only small amounts of DNA. However, see the next question for conformation issues.

Yes. We have seen a 20–30% difference. For the different forms of plasmid DNA, we recommend using a standard that more closely represents the composition of the plasmid DNA in the sample. For information on creating custom optimized assays, please visit our website.

PicoGreen® dye and other fluorescence-based quantification reagents are not recommended for quantifying dye-conjugated nucleic acids. The attached dye molecules can interfere with either binding and/or fluorescence output of the quantification reagents.

The buffer included in the kit should assure the proper pH range, even if your DNA is at a pH outside of this range, since at least a 10-fold excess of kit buffer over sample is used in the assay.

Yes, the manual has directions for this application. You will use the 0 ng/μL lambda dsDNA HS standard to generate Standard #1. You will prepare a dilution of the 10 ng/μL lambda dsDNA HS standard to generate Standard #2. You then prepare the samples and compare them to this 2-point standard curve. The Quant-iT dsDNA BR Kit can be used in a similar manner.

There are several differences:

• The Quant-iT™ RNA Assay uses a far-red dye (excitation 644 nm/emission 673 nm); the Quant-iT™ RiboGreen® RNA assay uses a green fluorescent dye (excitation 500 nm/emission 525 nm).
• The Quant-iT™ RNA assay is more specific for RNA.
• Quant-iT™ RNA Assays are less sensitive than the Quant-iT™ RiboGreen® RNA Assay.

The linear ranges are:

• Quant-iT™ RiboGreen® RNA Assay: 1 ng/mL to 1 µg/mL
• Quant-iT™ RNA BR Assay: 20–1,000 ng
• Quant-iT™ RNA HS Assay: 5–100 ng

Yes, the tag does not interfere with the signal.

No, they are not accurate for such small fragments. The Quant-iT™ microRNA Assay Kit and Qubit® microRNA Assay Kit are good tools for this application.

Yes, the manual has directions for this application.

No. The Qubit RNA IQ Assay does not assay the purity of the RNA sample, only the extent of degradation of the RNA (small versus large strands).

At present, we do not have a fluorescence-based assay that tests for sample purity (protein contamination). We do offer various instruments that can obtain absorbance readings (absorbance at 260/280), such as our NanoDrop, BioMate and GENESYS spectrophotometers.

No. The presence of an excess amount of free nucleotides/bases may affect the assay results in the same manner as other contaminants, but the reagents in this kit do not give a fluorescence response to nucleotides or bases.