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S U P P O R T
 

Protocol for DNA Digestion and Subsequent Dephosphorylation
(with Shrimp Alkaline Phosphatase (SAP))

  1. Prepare the following reaction mixture (final volume 20-50 µl) containing:
    1-3 µg DNA
    1X restriction enzyme buffer
    2 u of restriction enzyme for 1 µg of DNA
  2. Incubate at 37°C for 1 hour.
  3. Add 1 unit of SAP per 1 picomole of DNA 5'-termini. Incubate at 37°C for 30 min.
  4. Stop reactions by heating at 65°C for 15 min or at 80°C for 20 min (if restriction enzyme can not be inactivated at 65°C).

Note
If DNA is digested with restriction enzyme PaeI (SphI), it is recommended to extract the reaction mixture with phenol/chloroform and precipitate DNA with ethanol. Then, DNA is dissolved in 1X SAP buffer and dephosphorylated.

References

  1. Nilsen, I.W., et al., Thermolabile alkaline phosphatase from Northern shrimp (Padalus borealis): protein and cDNA sequence analyses, Comparative Biochemistry and Physiology, B, 129, 853-861, 2001.
  2. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
  3. Werle, E., et al., Convenient single-step, one tube purification of PCR products for direct sequencing, Nucleic Acids Res., 22, 4354-4355, 1994.
  4. Khosravi, R., et al., Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage, Proc. Natl. Acad. Sci USA, 96, 14973-14977, 1999.
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Updated sausio 31, 2008 16:28