Alkaline phosphatase, Escherichia coli

Instructions
1. Dissolve the enzyme in 30 U/μL 10 mM Tris-HCl (pH8.0), 1 mM MgCl₂, 50 mM KCl, 0.1 mM ZnCl₂. Fresh preparation is recommended.
2. Add the following reagents to a 1.5 mL centrifuge tube to dephosphorylate DNA:
1) 1-20 pmol DNA fragment
2) 1-2 μL Alkaline Phosphatase
3) 5 μL 500 mM Tris-HCl pH 9.0, 10 mM MgCl2
4) Add sterile ddH₂O to 50 μL
Note: Single-stranded DNA or DNA with 5' protruding ends is recommended to be incubated at low temperature, while blunt-ended DNA or DNA with 3' protruding ends is recommended to be incubated at higher temperature.
3. Mix well and incubate at 37°C or 50°C for 30 min. Alternatively, incubate at 37°C for 15 min, then incubate at 50°C for 15 min.
4. Extract twice with phenol/chloroform/isoamyl alcohol (25:24:1).
Note: Before phenol extraction, it is recommended to incubate it at 56°C for 30 min in a solution containing 5 mM EDTA, 0.5% SDS, and 50 μg/mL Proteinase K to completely inactivate alkaline phosphatase. Incubate at 75°C for 10 min before phenol extraction for better inactivation. 5. Extract with chloroform/isoamyl alcohol (24:1).
6. Add 2.5 μL 3 M NaCl (final concentration 150 mM), 125 μL (2.5 times volume) pre-cooled ethanol, mix well, and place at -20°C for 30-60 min to precipitate DNA.
4 Centrifuge, completely remove ethanol, and dissolve DNA in an appropriate amount of deionized water (<20 μL).

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.