36
KAI-OLAF NETZER
Subprotocol 3
Phosphatases - CIP Reaction
Two enzymes, bacterial alkaline phosphatase (BAP) and calf intestine alkaline phosphatase ( CIP) are used in molecular biology. The enzymes catalyze
the removal of 5' phosphate residues from DNA, RNA, rNTPs, and dNTPs.
The bacterial enzyme BAP is more active but is also far more resistant to
inactivation procedures at the end of dephosphorylation reactions.
Phosphatases are used to dephosphorylate vector DNA after restriction
enzyme digestion to prevent self ligation. It is essential, however, to completely remove the phosphatases so that subsequent ligations work efficiently. Another use is to remove 5' phosphates from DNA or RNA prior
to labeling 5' termini with 32 P.
Selection of phosphatase is mainly dependent on individual preference.
Both enzymes CIP and BAP work well. W e prefer calf intestinal phosphatase
since it is easier and less time consuming to remove after the reaction is
complete.
Materials
Supplies • Heating block (37°C)
• Microfuge
• Spin columns
Reagents • DNA tobe dephosphorylated (0.1-0.2Jlg/Jll in water, TE, or any restriction enzyme buffer containing 50 mM Tris-HCl)
• lOx CIP buffer (1 mM ZnCh, 10 mM MgC12, 500 mM Tris-HCl pH 8.0)
• CIP enzyme (1 U/J.!l; if purchased at higher concentration, dilute an aliquot in storage buffer to 1 U/J.!l, store at +4oC)
• EDTA 0.5 M pH 8.0
• Chaotropic buffer ( e. g. Qiaex™)
KAI-OLAF NETZER
Subprotocol 3
Phosphatases - CIP Reaction
Two enzymes, bacterial alkaline phosphatase (BAP) and calf intestine alkaline phosphatase ( CIP) are used in molecular biology. The enzymes catalyze
the removal of 5' phosphate residues from DNA, RNA, rNTPs, and dNTPs.
The bacterial enzyme BAP is more active but is also far more resistant to
inactivation procedures at the end of dephosphorylation reactions.
Phosphatases are used to dephosphorylate vector DNA after restriction
enzyme digestion to prevent self ligation. It is essential, however, to completely remove the phosphatases so that subsequent ligations work efficiently. Another use is to remove 5' phosphates from DNA or RNA prior
to labeling 5' termini with 32 P.
Selection of phosphatase is mainly dependent on individual preference.
Both enzymes CIP and BAP work well. W e prefer calf intestinal phosphatase
since it is easier and less time consuming to remove after the reaction is
complete.
Materials
Supplies • Heating block (37°C)
• Microfuge
• Spin columns
Reagents • DNA tobe dephosphorylated (0.1-0.2Jlg/Jll in water, TE, or any restriction enzyme buffer containing 50 mM Tris-HCl)
• lOx CIP buffer (1 mM ZnCh, 10 mM MgC12, 500 mM Tris-HCl pH 8.0)
• CIP enzyme (1 U/J.!l; if purchased at higher concentration, dilute an aliquot in storage buffer to 1 U/J.!l, store at +4oC)
• EDTA 0.5 M pH 8.0
• Chaotropic buffer ( e. g. Qiaex™)