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S U P P O R T
 

Protocol for Labeling DNA 3'-termini
(with Terminal Deoxynucleotidyl Transferase)

  1. Prepare the following reaction mixture:
    5X reaction buffer for Terminal Deoxynucleotidyl Transferase 10 µl
    DNA fragments 10 pmol of 3'-termini
    [alfa-32P]-ddATP, ~110 TBq/mmol (3000 Ci/mmol) 1.85 MBq (50 µCi)
    Terminal Deoxynucleotidyl Transferase 2 µl (40 u)
    Water, nuclease-free  to 50 µl
  2. Incubate the mixture at 37°C for 15 minutes.
  3. Stop the reaction by heating at 70°C for 10 minutes or by the addition of 5 µl 0.5M EDTA (pH 8.0).

Note
The efficiency of the reaction depends upon the type of 3'-OH termini of the DNA fragments. 3'-protruding ends are labeled with higher efficiency than recessed or blunt ends.

References

  1. Deng, G.R., Wu, R., Terminal transferase: Use in the tailing of DNA and for in vitro mutagenesis, Meth. Enzymol., 100, 96-116, 1983.
  2. Tu, C.-P.D., Cohen, S.N., 3'-end labeling of DNA with [alfa-32P]cordycepin-5'-triphosphate, Gene, 10, 177-183, 1980.
  3. Vincent, C., et al., Synthesis of 8-(2,4-dinitrophenyl-2,6-aminohexyl)aminoadenosine-5'-triphosphate: Biological properties and potential uses, Nucleic Acids Res., 10, 6787-6796, 1982.
  4. Kumar, A., et al., Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase, Anal. Biochem., 169, 376-382, 1988.
  5. Gaastra, W., Klemm, P., Radiolabeling of DNA with terminal transferase, Methods in Molecular Biology, vol.2: Nucleic Acids (Walker, J.M., ed.), Humana, Clifton, NJ, 269-271, 1984.
  6. Igloi, G.L., Schiefermayr, E., Enzymatic addition of fluorescein- or biotin-riboUTP to oligonucleotides results in primers suitable for DNA sequencing and PCR, BioTechniques, 15, 486-497, 1993.
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Updated gegužės 09, 2007 17:25