The classical method for purifying andor concentrating DNA is by alcohol

The classical method for purifying andor

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The classical method for purifying and/or concentrating DNA is by alcohol precipitation (1), which is frequently preceded by phenol/chloroform extraction (2, 3). However, silica adsorption and gel filtration are two popular techniques that are currently used for cleanup of reaction mixtures containing PCR fragments or restricted DNAs. Silica-based DNA cleanup products bind DNA in the presence of high concentrations of chaotropic salts and certain pH conditions, while contaminants such as primers, dNTPs, enzyme, and buffer salts are washed away. Silica adsorption is the most highly recommended method for DNA cleanup from PCR amplifications, agarose gel slices, and enzymatic reactions because it removes enzyme from the reaction mixture and can also be used to concentrate DNA. In addition, silica may be used to purify fluorescent-labeled DNA (e.g., Cy3- or Cy5-labeled cDNA). Silica is typically provided as a solid phase such as a membrane or filter, both of which are amenable to increased sample throughput.
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28-9624-00 AA 107 Gel filtration products such as Sephadex™ or Sephacryl™ in a microcentrifuge-based column format can be used to purify PCR products or DNA of interest directly from reaction mixture contaminants in approximately 5 min. The contaminants, which are smaller than the DNA product, are retained by the gel matrix while the DNA product is not. Sephadex G-25 and G-50 are typically used to remove unincorporated nucleotides and primers from reaction mixtures. Note that enzyme is not removed using the gel filtration method, which is discussed in more detail later in this chapter. Considerations for gel filtration in a spin format are discussed later in this chapter. An alternative option for PCR cleanup prior to sequencing is enzymatic treatment. ExoSAP-IT from GE Healthcare uses a mixture of two enzymes to degrade unincorporated nucleotides and primers. Figure 8.1 provides an overview of the ExoSAP-IT method. Additional details on ExoSAP-IT are provided in Chapter 9. PCR Add ExoSAP-IT 37°C, 15 min (treatment) 80°C, 15 min (inactivation) Sequencing reaction Exonuclease I +pN shrimp alkaline phosphatase 5’ 3’ OH 5’ 3’ OH N ExoSAP-IT Principle Fig 8.1. ExoSAP-IT enzymatic method for PCR product cleanup. N = nucleotides. Cleanup of DNA fragments from agarose gels For some applications, it may be critical for the fragment of interest to be free of competing, contaminating fragments. In such cases, removal of competing fragments can be accomplished by agarose (or polyacrylamide) gel electrophoresis (2). A gel slice containing the fragment of interest can be excised and the fragment extracted using a variety of methods. Electrophoresis is not only a method for analysis of nucleic acids; it is also a valuable preparative technique. The various electrophoretic separation techniques will not be considered here.
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  • Spring '12
  • CKwong
  • DNA, GE Healthcare

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