An optimized PCR-based procedure for production of 13C/15N-labeled DNA.
We have substantially improved a procedure that we previously described for producing 13C/15N-labeled DNA (Chen et al., FEBS Lett. 436, 372-376, 1998) to provide an economical and straightforward approach to the preparation of labeled DNA. The conditions for the PCR reactions have been optimized to permit the use of low concentrations of the costly labeled dNTPs (50 microM for each). In addition, a rapid and high-yield purification procedure has been developed that allows us to obtain a high yield of very pure labeled DNA. These modifications to our original procedure permit us to obtain 1.9 mg of an 18 bp DNA oligomer from 20 mg of dNTPs (ca. 10% yield from the starting dNTPs). This is sufficient material for the preparation of 0.4 mM sample in a volume of 400 microl. In summary, this procedure is a cost-effective, time-efficient procedure for the production of labeled DNA for NMR studies.
Yan
J
J
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908-0736, USA.
Bushweller
J H
JH
D002247:Carbon Isotopes / Q000032:analysis / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D007553:Isotope Labeling / Q000379:methods*; D009363:Neoplasm Proteins; D009587:Nitrogen Isotopes / Q000032:analysis / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D016133:Polymerase Chain Reaction / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism
D002247:Carbon Isotopes; D017931:DNA Primers; D009363:Neoplasm Proteins; D009587:Nitrogen Isotopes; D014157:Transcription Factors; D004247:DNA