Protocol for Dephosphorylation of 5´-ends of DNA using rSAP (M0371)
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- Prepare a 20 μl reaction as follows:
DNA 1 pmol of DNA ends* rCutSmart™ Buffer (10X) 2 µl rSAP 1 unit Nuclease-free water to 20 µl**
- Incubate at 37°C for 30 minutes.
- Stop reaction by heat-inactivation at 65°C for 5 minutes.
**Scale larger reaction volumes proportionally.
Dephosphorylation of 5´- ends of DNA in Restriction Enzyme Reaction
- The phosphatase can be added directly into the digestion reaction during or after DNA digestion
- rSAP is active in all NEB restriction enzyme buffers
- The restriction enzyme should be heat inactivated at the same time as the phosphatase after digest and dephosphorylation
- If restriction enzyme(s) cannot be heat inactivated, DNA purification is required before ligation