General description
Application
Deoxyribonucleic acid, single stranded from salmon testes has been used for/as:
- in situ hybridization histochemistry of rat brain, human kidney sections and renal biopsy tissues
- preparing standard curve of DNA
- prehybridization solution in southern and northern hybridizations
- dilution of the cDNA
- fluorescent in-situ hybridization (FISH)
- ChIP analysis
- a standard to analyse the degraded nucleosides of genomic DNA by high performance liquid chromatography
- a component of hybridization solution in FISH
- a control in DNA-DNA hybridization experiment between various bacterial species
- to precipitate probes in fluorescence in situ hybridization (FISH)
Many factors contribute to the signal-to-noise ratio in nucleic acid hybridizations. These factors include the presence of solvent (formamide), hybridization temperature, length of hybridization, volume of hybridization solution, degree and method of agitation, use of blocking reagents, concentration and specific activity of the probe, use of molecular agents to increase the rate of nucleic acid reassociation, and the degree of stringency used during the washing of the membrane.
In order to decrease any non-specific hybridization of the probe to a substrate, blocking agents must be used. Generally, a combination of blocking reagent, detergent, and denatured, fragmented DNA is used to accomplish this. Sigma offers sonicated, denatured DNA from a variety of species for use as a blocking agent in Northern and Southern blotting and other nucleic acid hybridization techniques.
Preparation Note
Other Notes
Platform
| Spectrofluorometer | |
| Excitation | 490 nm |
| Emission | 525 nm |
| Cutoff | 515 nm |
Components
| Component A: Helixyte Green™ | 1 vial (1 mL, 200X in DMSO) |
| Component B: 20X Assay Buffer | 1 bottle (25 mL) |
| Component C: Calf thymus DNA Standard | 1 vial (1 mL, 100 µg/mL) |
Example protocol
At a glance
Protocol summary
- Add 1mL dsDNA standards or test samples to each cuvette
- Add 1mL Helixyte Green™ working solution
- Incubate at RT for 5-10 minutes
- Monitor the fluorescence at Ex/Em=490/525 nm
Important notes
The following protocol is an example for quantifying dsDNA with Helixyte Green™. Allow all the components to warm to room temperature before opening. No data is available addressing the mutagenicity or toxicity of Helixyte Green™dsDNA stain. Because this reagent binds to nucleic acids, it should be treated as a potential mutagen and handled with appropriate care. The DMSO stock solution should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues.
Preparation of stock solution
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
1. Assay Buffer (1X)
Prepare a 1X Assay buffer by diluting the concentrated buffer 20-fold with sterile, distilled, DNase-free water.
Preparation of standard solution
For convenience, use our Serial Dilution Planner
For high range standard curve:
Add 30 µL of 100 µg/mL dsDNA stock solution (Component C) to 1.47 mL of 1X Assay buffer to have 2000 ng/mL dsDNA solution, and then perform 1:2 and 1:10 serial dilutions to get 1000, 100, 10, 1 and 0 ng/mL.
For low range standard curve:
Add 40 µL of 2 µg/mL dsDNA stock solution to 1.56 mL of 1X Assay buffer to have 50 ng/mL dsDNA solution, and then perform 1:2 and 1:10 serial dilutions to get 25, 2.5, 0.25, 0.025 and 0 ng/mL.
Preparation of working solution
Prepare Helixyte Green™ working solution by making a 200-fold dilution of the concentrated DMSO solution in 1X assay buffer. For example, to prepare enough working solution to assay 10 samples in a 2 mL final volume, add 50 μL of Helixyte Green™ (Component A) into 10 mL of Assay Buffer (Component B). Protect the working solution from light by covering it with foil or placing it in the dark. Note: We recommend preparing this solution in a plastic container rather than glass, as the dye may adsorb to glass surfaces. For best results, this solution should be used within a few hours of its preparation.
Procedure
- Add 1 mL of Helixyte Green™ working solution to each cuvette containing 1 mL of the dsDNA standard, blank control, and test samples to make the total dsDNA assay volume of 2 mL/cuvette.
- Incubate the reaction at room temperature for 5 to 10 minutes, protected from light.
- Monitor the fluorescence increase with a spectroflurometer at Ex/Em = 490/525 nm. Note: To minimize photobleaching effects, keep the time for fluorescence measurement constant for all samples.
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Product name
DNA Quantification Assay Kit (Fluorometric) -
Detection method
Fluorescent -
Sample type
Tissue, Adherent cells, Suspension cells, Whole Blood, Purified mitochondria -
Sensitivity
4 ng/ml -
Range
0.4 ng – 150 ng -
Product overview
DNA Quantification Assay Kit (Fluorometric) (ab252898) provides a quick, specific, and easy method for the measurement of DNA concentrations in a wide variety of samples. In this assay, DNA specifically reacts with a probe producing a stable fluorescence signal (Ex/Em = 492/528 nm). The kit is simple to perform, specific, sensitive and high-throughput adaptable with a wide detection range (0.4-150 ng DNA) and a limit of detection of 4 pg/µL dsDNA in samples.
