Engineering:GeneQuant
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GeneQuant Spectrophotometer is an RNA/DNA calculator tool that measures absorbance at 230, 260, 280, 320, 546, 562, 595 and 600 nm. It can determine the concentration and purity of nucleic acids after PCR amplification, calculate annealing temperatures for primers prior to PCR upon entry of the oligonucleotide sequence and concentration and the total buffer molarity, measure Optical Density (OD) 600 for bacterial cell culture solutions, and it may be used for protein determination using the Bradford, Biuret, and BCA protein assays.[1]
Techniques
Nucleic Acid Quantification (NAQ)
A solution of DNA or RNA with an optical density of 1.0 has a concentration of 50 or 40 ug/mL, respectively, in a 10 mm pathlength cell. Oligonucleotides have a corresponding factor of 33 ug/mL which can be calculated if the base sequence is known.[2]
Concentration = Abs260 * Factor
The instrument uses factors 50, 40 and 33 as defaults for RNA, DNA, and Oligonucleotides, respectively, and compensates for dilution and use of calls which do not have 10 mm pathlength.
Default units are converted using: 1 ug/mL = 1 ng/uL = 0.001 ug/uL pmol/uL = (ug/mL * 1000)/ MW of oligo pmol phosphate = (nucleotide concentration, ug/mL) / 315
Molecular weight of a DNA oligonucleotide is calculated: MW (g/mole) = [(dA * 312.2) + (dC * 288.2) + (dG * 328.2) + (dT * 303.2)] + [(MW counter-ion) * (length of oligo in bases)]
- For RNA nucleotide, (dT * 303.2) is (dU * 298.2)
The MW is calculated using the following equation and adjusted for the contribution of the atoms at 5' and 3' ends: Phosphorylated oligos add: [17 + (2 * MW counter-ion)] Non-phosphorylated add: [61 + (MW counter-ion)] MW (g/mole) of the most common counter-ions are NA, 23.0, K, 39.1, and TEA, 102.2.
References
- ↑ "404 Not Found - GE Healthcare Life Sciences". https://www.gelifesciences.com/gehcls_images/GELS/Related+Content/Files/1314750913712/litdoc28925114AA_20110831033722.pdf.[|permanent dead link|dead link}}]
- ↑ "404 Not Found - GE Healthcare Life Sciences". https://www.gelifesciences.com/gehcls_images/GELS/Related+Content/Files/1314723116657/litdocGeneQuantpro_20110830203746.pdf.[|permanent dead link|dead link}}]
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