R.W., D. Botstein and J.R. Roth, A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. 1980.] B. Purification To remove protein from nucleic acid solutions: 1. Treat with proteolytic enzyme, e.g., pronase, proteinase K 2. Purify on a silica-based column such as a Qiagen PCR Prep Column 3. CsCI/ethidium bromide density gradient 4. Phenol Extract. The simplest method for purifying DNA is to extract with phenol or phenokchloroform and then chloroform. The phenol denatures proteins and the final extraction with chloroform removes traces of phenol 5. Purify on silica-based column such as Qiagen Brand columns (http://www.qiagen.com) C. Quantitation . 1. Spectrophotometric. For pure solutions of DNA, the simplest method of quantitation is reading the absorbance at 260 nm where an OD of 1 in a 1 cm path length = 50 u g/ml for double-stranded DNA, 40 u g/ml for single- stranded DNA and RNA and 20-33 u g/ml for oligonucleotides. An absorbance ratio of 260 nm and 280 nm gives an estimate of the purity of
R.W., D. Botstein and J.R. Roth, A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. 1980.] B. Purification To remove protein from nucleic acid solutions: 1. Treat with proteolytic enzyme, e.g., pronase, proteinase K 2. Purify on a silica-based column such as a Qiagen PCR Prep Column 3. CsCI/ethidium bromide density gradient 4. Phenol Extract. The simplest method for purifying DNA is to extract with phenol or phenokchloroform and then chloroform. The phenol denatures proteins and the final extraction with chloroform removes traces of phenol 5. Purify on silica-based column such as Qiagen Brand columns (http://www.qiagen.com) C. Quantitation . 1. Spectrophotometric. For pure solutions of DNA, the simplest method of quantitation is reading the absorbance at 260 nm where an OD of 1 in a 1 cm path length = 50 u g/ml for double-stranded DNA, 40 u g/ml for single- stranded DNA and RNA and 20-33 u g/ml for oligonucleotides. An absorbance ratio of 260 nm and 280 nm gives an estimate of the purity of