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S U P P O R T
 

Protocols for DNA Labeling by Nick-translation
(with DNA Polymerase I, E.coli):

Nick-translation using Radioactive Nucleotides

  1. Mix the following components:
    10X reaction buffer for DNA Polymerase I 2.5 µl
    mixture of 3 dNTPs, 1 mM* (without the labeled dNTP) 1.25 µl
    [alfa-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol) 1.85-3.7 MBq (50-100 µCi)
    DNase I, RNase-free
    freshly diluted to 0.002 u/µl**     		1 µl
    DNA Polymerase I, E.coli 0.5-1.5 µl (5-15 u)
    template DNA 0.25 µg
    Water, nuclease-free to 25 µl
  2. Immediately incubate at 15°C for 15-60 minutes.
  3. Terminate the reaction by adding 1 µl of 0.5M EDTA, pH 8.0.
  4. Take an aliquot (1 µl) to determine efficiency of the label incorporation. A specific activity of  DNA at least 108 cpm/µg  DNA is expected.
  5. If needed, the labeled DNA may be separated from the unincorporated radioactive precursors on Sephadex G-50 or Bio-Gel P-60 column.

Note

* To prepare a mixture of three non-labeled dNTPs (1 mM of each), mix 1 µl aliquots of stock solutions of each dNTP (100 mM, from #R0181) with 97 µl of Water, nuclease-free. These dNTP mixes can be stored at -20°C for further use.
** The DNase I, RNase-free can be diluted with the 1X reaction buffer for DNA Polymerase I.

Nick-translation using Biotin-11-dUTP

  1. Mix the following components:
    10X reaction buffer for DNA Polymerase I 2.5 µl
    mixture of 3 dNTPs, 1 mM* (without the labeled dNTP) 1 µl
    1 mM dTTP 0.2 µl
    1 mM Biotin-11-dUTP 1 µl
    DNase I, RNase-free
    freshly diluted to 0.002 u/µl**     		1 µl
    DNA Polymerase I, E.coli 0.5-1.5 µl (5-15 u)
    template DNA 0.25-0.5 µg
    Water, nuclease-free to 25 µl
  2. Immediately incubate at 15°C for 1-2 hours.
  3. Place the reaction tube on ice.
  4. Electrophorese 3 µl aliquots of the reaction mixture on agarose gel. Use the appropriate Fermentas DNA ladder to evaluate the size of the labeled probe. The optimal size of the probe is 200-500 bp. If the probe is longer, continue incubation at 15°C until DNA is trimmed to the proper size.
  5. Terminate the reaction by adding 1 µl of 0.5M EDTA, pH 8.0.

Note

* To prepare a mixture of three non-labeled dNTPs (1 mM of each), mix 1 µl aliquots of stock solutions of each dATP, dCTP, dGTP (100 mM, from #R0181) with 97 µl of Water, nuclease-free. These dNTP mixes can be stored at -20°C for further use.
** The DNase I, RNase-free can be diluted with the 1X reaction buffer for DNA Polymerase I.

References

  1. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Brooklyn, New York, 3.5.3-3.5.6., 1994-2004.
  2. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Horbor laboratory, Cold Spring Harbor, N. Y., 2001.
  3. Yu, H., et al., Cyanine dye dUTP analogs for enzymatic labeling of DNA probes, Nucleic Acids Res., 22, 3226-3232, 1994.
  4. Gubler, U., Hoffmann, B.J., A simple and very efficient method for generating cDNA libraries, Gene, 25, 263-269, 1983.
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Updated vasario 05, 2008 10:26