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Protocol for Fast Simultaneous Plasmid Vector Linearization and Dephosphorylation
(with FastAP™ Thermosensitive Alkaline Phosphatase)

1. Prepare the following reaction mixture:
   

   Plasmid DNA	1 µg
   10X FastDigest® buffer	2 µl
   FastDigest® restriction enzyme	1 µl
   FastAP™ Thermosensitive Alkaline Phosphatase	1 µl (1 u)
   Water, nuclease-free	to 20 µl

2. Mix thoroughly, spin briefly and incubate at 37°C for 10 minutes.
3. Stop reactions by heating at 65°C for 15 minutes or at 80°C for 20 minutes (if restriction enzyme is not inactivated at 65°C).

Note
For FastDigest® SphI (PaeI), simultaneous digestion and dephosphorylation is not recommended. Perform digestion, phenol/chloroform extract and ethanol precipitate digested DNA, dissolve DNA in 1X FastDigest® buffer and dephosphorylate.

Ordering Information

FastAP™ Thermosensitive Alkaline Phosphatase FastDigest® restriction enzymes Water, nuclease-free

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Updated sausio 31, 2008 08:53