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DNA > DNA MODIFYING ENZYMES

S1 Nuclease Digest of ssDNA

   

Alkaline Phosphatase Digestion of DNA

   

BAP

    CIP

BAP

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To prepare BAP from the ammonium sulfate suspension its sold as, spin out the precipitate for 5 min at 4C in microfuge, resuspend in H2O to a concentration of 0.1 units/ul, and centrifuge desalt on Sephadex G25 equilibrated in H2O. Adjust the desalted BAP with Tris pH 8 to 0.1 M and store at 4 C stable essentially indefinitely.

To 10 ug DNA linearized plasmid or fragments dissolved in 45 ul H2O. Add 5 ul CB 10 X BAP, preheat to 68C and add 1 ul BAP (0.1 units/ul Worthington). Incubate at 68C for 1/2 hr, add another aliquot of BAP (1 ul) and continue incubation for another 1/2 hr. Then add phenol and chloroform directly to 68C reaction volume and vortex, do multiple extractions, add NaCl to 0.2 M and ethanol precipitate as usual.

CB 10 X BAP
Tris 0.5 M pH 8.0
MgCl2 20 mM
ZnCl2 1 mM