The Qubit 2.0 Fluorometer
The Qubit dsDNA HS (High Sensitivity) Assay Kit is designed specifically for use with the Qubit Fluorometer. The assay is highly selective for double-stranded DNA (dsDNA) over RNA and is designed to be accurate for initial sample concentrations from 10 pg/µL to 100 ng/µL. The kit provides concentrated assay reagent, dilution buffer. DNA Quantitation Methods: Evaluation and Comparison. Participants: Margaret C. Kline, Amy E. Decker, and Peter M. Project Timeframe: 2004 to present Purpose: Real-time quantitative PCR (qPCR) has generated a great deal of interest in the forensic DNA typing community in the past several years as this technique can rapidly detect low levels of DNA with minimum hands-on time and minor.
TheQubit fluorometeris a laboratory instrument created and dispersed by Invitrogen that, among some other applications, will be utilized for the quantification of DNA, RNA, and proteins.1234
Processedit
The Qubit fluorometer utilizes fluorescent dyes to determine the focus of either nucleic acids or protein in a small sample. The UV-absorbance technique utilizes a spectrophotometer to calculate the organic absorbance of lighting at 260 nm (for DNA and RNA) or 280 nm (for proteins). The more DNA, RNA or proteins in the example, the more light is assimilated at this wavelength. The absorbance is certainly a organic real estate of DNA, RNA, free of charge nucleotides, proteins and some amino acids and numerous other compounds as properly. Because therefore many elements absorb light at 260 nm, this dimension is subject to inaccuracy due to possible contamination of the structure with these some other substances. In addition, making use of the absorbance method, it is not feasible to differentiate between DNA, RNA, protein or free of charge nucleotides or amino acids in the structure, leading to possibly highly inaccurate measurements.5678
Fluorescent dyesedit
The Qubit assays (previously identified as Quant-iT) had been developed and manufactured by the previous Molecular Probes (now a part of Life Systems). Each coloring is specific for one type of molecule (DNA, RNA or protein). They have extremely reduced fluorescence until guaranteed to their focus on molecule. The distinction in fluorescence between bound and unbound coloring is various orders of size. Upon binding to DNA, most likely by intercalation between the angles, it takes on a even more rigid shape and gets to be intensely fluorescent.910As soon as included to a option of DNA, the Qubit DNA dye binds to the DNA within secs and reaches balance in much less than two moments.
At a particular amount of the color, the quantity of fluorescence indication from this blend is straight proportional to the concentration of DNA in the option, also in the existence of various other bio-molecules. The Qubit fluorometer picks this fluorescence signal up and changes it into a DNA concentration measurement by referring to DNA probes of identified focus. It after that uses this connection to calculate the concentration of a structure.
Qubit 2.0 fluorometer with the 'dsDNA BR Assay Package'
The Qubit quantitation system contains the adhering to dyes that are particular for different bio-molecules and levels (ds stands for double-stranded, ss for single-stranded DNA):
Reagent/Assay | Assay variety | Structure starting focus variety |
---|---|---|
Qubit dsDNA HS Assay | 0.2-100 ng | 10 pg/μd-100 ng/μd |
Qubit dsDNA BR Assay | 2-1,000 ng | 100 pg/μl-1 μgary the gadget guy/μl |
Qubit ssDNA Assay | 1-200 ng | 50 pg/µM-200 ng/µL |
Qubit RNA Assay | 5-100 ng | 250 pg/μd-100 ng/μm |
Qubit RNA BR Assay | 20-1,000 ng | 1 ng/µ-1 µgary the gadget guy/µL |
Qubit Proteins Assay. | 0.25-5 μg | 12.5 μgary the gadget guy/ml-5 mg/ml |
Comparison with other devicesedit
Various other fluorometers can also determine the fluorescence from the Qubit dyes and can end up being used for DNA, RNA and protein quantification in the exact same way. However, all additional fluorometers require the user to make use of many DNA criteria and plot the concentration versus the absorbance on a chart. The information must after that be installed to a line and lastly the example concentration calculated from the formula of the collection. Although this can be a simple computation for any scientist, the Qubit fluorometer will this calculation for the consumer, producing it faster and less complicated, in addition to becoming less expensive than a regular fluorometer.citation needed
Versionsedit
The second generation, the Qubit 2.0 Fluorometer, has been launched in 2010, the 3rchemical era as Qubit 3.0 in 2014. The newest version can be Qubit 4 which was launched in 2017.
Personal referencesedit
- ^Acar E, et al. (2009). 'Optimization and affirmation studies of the MentypeR Argus Back button-8 package for paternity instances'.Forensic Sci Int Genet Suppl.2: 47-48. doi:10.1016/l.fsigss.2009.08.189.
- ^Bakos L, et al. (2009). 'Overflowing environment affects hormonal standing and hippocampal mind produced neurotrophic factor in a sex dependent manner'.Neuroscience.164(2): 788-797. doi:10.1016/m.neuroscience.2009.08.054. PMID19723563.
- ^Halaihel N, et al. (2009). 'A new real time PCR-based assay for diagnosing Renibacterium salmoninarum in rainbow bass (Oncorhynchus mykiss) and comparison with various other techniques'.J Microbiol Meth.76(1): 75-80. doi:10.1016/l.mimet.2008.09.014. PMID18938198.
- ^Hamza IA, et al. (2009). 'Recognition and quantification of individual bocavirus in riverwater'.M Gen Virol.90(Pt 11): 2634-2637. doi:10.1099/vir.0.013557-0. PMID19656966.
- ^Manchester, K.L. (1996). 'Make use of of UV methods for the measurement of protein and nucleic acid levels'.BioTechniques.20(6): 968-970. PMID8780864.
- ^Glasel, J.A new. (1995). 'Validity of nucleic acid purities monitored by 260 nm/280 nm absorbance ratios'.BioTechniques.18(1): 62-63. PMID7702855.
- ^Huberman, M.A new. (1995). 'Importance of measuring nucleic acid absorbance at 240 nm mainly because well as at 260 and 280 nm'.BioTechniques.18(4): 636. PMID7598897.
- ^Manchester, E.M. (1995). 'Worth of A260/A280 proportions for measurement of purity of nucleic acids'.BioTechniques.19(2): 208-210. PMID8527139.
- ^McKnight, R.E., Gleason, A.C., Keyes, M.A., Sahabi, S i9000. (2006). 'Presenting setting and affinity studies of DNA-binding real estate agents making use of topoisomerase I DNA unwinding assay'.Bioorganic amplifier; Medicinal Hormone balance Letters.17(4): 1013-1017. doi:10.1016/m.bmcl.2006.11.038. PMID17157016.CS1 maint: Several brands: authors list (hyperlink)
- ^Schweitzer, Chemical., Scaiano, J.M. (2003). 'Selective binding and regional photophysics of the fluorescent cyanine dye PicoGreen in double-stranded and single-stranded DNA'.Physical Chemistry Chemical substance Physics.5: 4911-4917. doi:10.1039/w305921a.CS1 maint: Multiple brands: writers list (hyperlink)
Outside linksedit
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