Attention:

Certain features of Sigma-Aldrich.com will be down for maintenance the evening of Friday August 18th starting at 8:00 pm CDT until Saturday August 19th at 12:01 pm CDT.   Please note that you still have telephone and email access to our local offices. We apologize for any inconvenience.

Alkaline Phosphatase Protocol & Troubleshooting

Product No. APMB-RO

Protocol

Biotinylated Alkaline Phosphatase

AP can be biotinylated using the Roche Biotin Protein Labeling Kit (Cat.No. 11418165001).

Troubleshooting

When a given nucleic acid is not dephosphorylated using the recommended reaction conditions, try using a higher Mg2+ concentration and spermidine. For 5 pmol of 5′-phosphate termini, dissolve the nucleic acid in 10 μl of 50 mM Tris-HCl/pH 8.0, containing 10 mM MgCl2, 0.5 mM spermidine, 0,01 mM EDTA and 0.1 mM ZnSO4 at room temperature (+15 to +25 °C). Do not place on ice, because spermidine-DNA complexes precipitate when cooled. Add 0.5 U AP (in 1 μl), and incubate the reaction mix at +37 °C for 30 min. Add a second aliquot of AP (0.5 U) and incubate further 30 min. To inactivate AP, heat the samples 20 min at +68 °C, add 1 μl 0.25 M EGTA (pH 7.8) and incubate a further 20 min at +68 °C. For these two heating steps, centrifuge the sample tubes briefly after each 10 min at +68 °C incubation, to remove condensation from the tube walls and prevent precipitation of spermidine-DNA complexes.

Materials

     
Related Links