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SPECTROPHOTOMETRIC ANALYSIS OF NUCLEIC ACIDS
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Objectives Learn how to do dilutions Use a spectrophotometer to calculate concentrations of nucleic acids Use absorbance data to figure out the purity of nucleic acids
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Spectroscopy Nucleic acids absorb light Best absorbance at 260nm
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Absorbance and Concentration Different molecules have different absorbance properties Absorbance reading of 1.0: dsDNA: 50 ug/ml ssDNA or RNA: 40 ug/ml Single stranded oligonucleotides: 33 ug/ml
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Absorbance and purity 280nm: absorbance peak for proteins A260 / A280 ratio for purity 1.8 to 1.9 indicate high quality DNA. Low ratios indicate proteins. 1.9 to 2.0 for RNA 230nm: absorbance peak for phenol A260 / A230 ratio for purity. The lower the less pure. A325: contamination from cuvette. Should be 0
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Dilutions Example: 1:20 dilution Total volume is 20 parts. 1 part of it is the sample (stock). The remaining 19 parts is water. Say you want the total volume to be 1ml (1000ul) Volume of sample = 1000 / number of parts. 1000 / 20 = 50 ul How much water? 1000 – 50 = 950 ul
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DNA Electrophoresis Objective: to run electrophoresis of PCR amplified Alu insert to determine genotype for Alu insertion to perform Hardy-Weinberg analysis of class result
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review mehmet - SPECTROPHOTOMETRIC ANALYSIS OF NUCLEIC...

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