ALKALINE PHOSPHATASE REMOVAL OF PO4 FROM DNA FRAGMENTS
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| PREPARE SOLUTIONS | |
| 1. 10X Dephosphorylation buffer: |
0.5 M Tris-HCl, pH 8.5, 1 mM EDTA
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| PROCEDURE | |
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NOTE: 1 unit of Alkaline Phosphatase (AP) can hydrolyze 50 pmol of 5'' terminal phosphorylated DNA fragments (3'' recessed, 5'' recessed or blunt-ended) when incubated at 37o C for 1 hour. This implies that 25 pmol of DNA are dephosphorylated (at both ends) in one hour by 1 unit of AP at 37o C |
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| This protocol takes into account the prior preparation of a DNA fragment, such as one isolated from LM agarose (DNA Fragment Isolation from LM agarose) | |
| 1. Phosphatase treat appropriate samples (for example: if digested vector can religate, i.e. digested by a single enzyme): mix 20 m L of 10X dephosphorylation buffer, 10 m L of Calf alkaline phosphatase (1U/m L) (contains 50% glycerol, so the volume of AP used should never be more than 10% of the final volume), and ~170 m L of Vector + dH2 O (fragment) (assuming ~0.5 m g) (200 m L total volume) | |
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2. Incubate at 37o C for 1 hour |
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| 3. Add 0.1X Vol of 200mM EDTA/EGTA (in this case, 20 m L) and HEAT inactivate at 65o C for >10 mins. The DNA is ready for downstream applications | |
| NOTE: the EDTA/EGTA should not interfere with ligation reactions if only 0.1X Vol of the AP reaction is used in the ligation reaction (no purification step in between, see DNA Ligation), in which case the EDTA/EGTA would have been dropped down to 1 to 2mM) | |
| NOTE: Note: when determining the amount of DNA to use: 1) determine concentration by spectrophotometer or better 2) run an aliquots (1, 5, 10 m L) on a gel. Determine the amount that is barely visible (EtBr has a visibility limit of about 100-200 ng on agarose gels) on a THIN gel (thick gels will mask some of the signal, specially at low DNA concentrations). Use two to three times more for the AP reaction. For example: if the aliquot that could be detected in the gel was 5 m L, then use 10 to 15 m L and dilute with dH2 O to the appropiate volume (in the example above, to 170 m L with 160 to 155 m L dH2 O) |
