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  • DNA Preparation and Quantitation (E2612)

    Note: It is important to use sterile technique to avoid environmental DNA contamination.

    Any method for the purification of protein-free genomic DNA can be used, including Proteinase K treatment followed by phenol/chloroform extraction and ethanol precipitation, lysozyme digestion, Qiagen® column preparation (for genomic DNA) or other methods. Sonication, nebulization, chaotropic salts, enzymatic fragmentation, rough handling, multiple freeze-thaws, or any other procedure that would cause DNA to shear, should be avoided. Samples should be in DNase-free TE buffer (pH 7.5) and be at least 15 kb or greater in size, and free of small molecular weight fragments. If the DNA fragment size is < 15 kb, enrichment will be less efficient. Determine DNA quality and quantity by agarose gel electrophoresis of the sample alongside a DNA marker (e.g., Lambda DNA-HindIII Digest, NEB #N3012). A sample of human (fetal lung fibroblast) IMR 90/E. coli (10:1 ratio) genomic DNA is included as a control. Generally, 5 μl of this DNA standard (0.5 μg) is sufficient to visualize on a 1% agarose gel alongside the experimental sample. It is also important to quantitate the amount of DNA in the experimental sample by A260 measurement using a spectrophotometer, such as a Nanodrop™ instrument.