religation of linearized plasmid DNA. The enzyme Reagents Supplied with Enzyme: Functional Assay: Dephosphorylation with CIP of Alkaline acts on 5´ protruding, 5´ recessed and blunt ends. 10X CutSmart™ Reaction Buffer a restriction enzyme-digested vector DNA with 5´ Phosphatase, CIP may also be used to degrade unincorporated recessed ends, the least favorable type for dephos- dNTPs in PCR reactions to prepare templates for Reaction Conditions: 1X CutSmart Reaction Buffer. phorylation, reduces re-ligation to < 0.5% compared Calf Intestinal (CIP) DNA sequencing or SNP analysis. Incubate at 37°C. to untreated control as measured by transformation 1-800-632-7799 into E. coli. CIP has been functionally tested in the [email protected] Source: Calf intestinal mucosa 1X CutSmart Reaction Buffer: following protocol: www.neb.com 50 mM Potassium acetate M0290S 064140416041 Specific Activity: ~ 3,000 units/mg 20 mM Tris-acetate Protocol for Dephosphorylation of 5´-ends of DNA 10 mM Magnesium acetate using CIP Molecular Weight: CIP is a homodimer. The 100 µg/ml BSA 1. Prepare a 20 μl reaction as follows: M0290S molecular weight of the monomer is 69 kDa. pH 7.9 @ 25°C DNA 1 pmol of DNA ends* 1,000 units Lot: 0641404 Exp: 4/16 Applications: Unit Definition: One unit is defined as the amount CutSmart Buffer (10X) 2 µl Dephosphorylation of cloning vector DNA to 10,000 U/ml Store at –20°C • of enzyme that hydrolyzes 1 µmol of p-Nitrophenyl CIP 1 unit prevent recircularization during ligation Phosphate, PNPP (NEB #P0757) in a total reaction Description: Alkaline Phosphatase, Calf H O, purified to 20 µl** • Dephosphorylation of DNA prior to end-label- volume of 1 ml in 1 minute at 37°C. 2 Intestinal (CIP) nonspecifically catalyzes the ing using T4 Polynucleotide Kinase dephosphorylation of 5´ and 3´ ends of DNA 2. Incubate at 37°C for 30 minutes. • Treatment of dNTPs in PCR reactions prior to Unit Assay Conditions: 1 M diethanolamine- and RNA phosphomonoesters. Also, CIP HCl (pH 9.8), 0.5 mM MgCl , 50 mM p-nitro- *Note: 1 pmol of DNA ends is about 1 µg of a 3 kb sequencing or SNP analysis 2 hydrolyses ribo-, as well as deoxyribonucleoside phenylphosphate and enzyme. These conditions are plasmid. Dephosphorylation of DNA and RNA triphosphates (NTPs and dNTPs). CIP is useful in • only used for quantitating enzyme activity. many molecular biology applications such as the **Scale larger reaction volumes proportionally. Supplied in: 50 mM KCl, 10 mM Tris-HCl (pH 8.2 removal of phosphorylated ends of DNA and RNA @ 25°C), 1 mM MgCl , 0.1 mM ZnCl and 50% 3. Purify DNA by gel purification, spin-column or for subsequent use in cloning or end-labeling of 2 2 glycerol. phenol extraction. probes. In cloning, dephosphorylation prevents

Now with CutSmart Reaction Buffer CERTIFICATE OF ANALYSIS

religation of linearized plasmid DNA. The enzyme Reagents Supplied with Enzyme: Functional Assay: Dephosphorylation with CIP of Alkaline acts on 5´ protruding, 5´ recessed and blunt ends. 10X CutSmart™ Reaction Buffer a restriction enzyme-digested vector DNA with 5´ Phosphatase, CIP may also be used to degrade unincorporated recessed ends, the least favorable type for dephos- dNTPs in PCR reactions to prepare templates for Reaction Conditions: 1X CutSmart Reaction Buffer. phorylation, reduces re-ligation to < 0.5% compared Calf Intestinal (CIP) DNA sequencing or SNP analysis. Incubate at 37°C. to untreated control as measured by transformation 1-800-632-7799 into E. coli. CIP has been functionally tested in the [email protected] Source: Calf intestinal mucosa 1X CutSmart Reaction Buffer: following protocol: www.neb.com 50 mM Potassium acetate M0290S 064140416041 Specific Activity: ~ 3,000 units/mg 20 mM Tris-acetate Protocol for Dephosphorylation of 5´-ends of DNA 10 mM Magnesium acetate using CIP Molecular Weight: CIP is a homodimer. The 100 µg/ml BSA 1. Prepare a 20 μl reaction as follows: M0290S molecular weight of the monomer is 69 kDa. pH 7.9 @ 25°C DNA 1 pmol of DNA ends* 1,000 units Lot: 0641404 Exp: 4/16 Applications: Unit Definition: One unit is defined as the amount CutSmart Buffer (10X) 2 µl Dephosphorylation of cloning vector DNA to 10,000 U/ml Store at –20°C • of enzyme that hydrolyzes 1 µmol of p-Nitrophenyl CIP 1 unit prevent recircularization during ligation Phosphate, PNPP (NEB #P0757) in a total reaction Description: Alkaline Phosphatase, Calf H O, purified to 20 µl** • Dephosphorylation of DNA prior to end-label- volume of 1 ml in 1 minute at 37°C. 2 Intestinal (CIP) nonspecifically catalyzes the ing using T4 Polynucleotide Kinase dephosphorylation of 5´ and 3´ ends of DNA 2. Incubate at 37°C for 30 minutes. • Treatment of dNTPs in PCR reactions prior to Unit Assay Conditions: 1 M diethanolamine- and RNA phosphomonoesters. Also, CIP HCl (pH 9.8), 0.5 mM MgCl , 50 mM p-nitro- *Note: 1 pmol of DNA ends is about 1 µg of a 3 kb sequencing or SNP analysis 2 hydrolyses ribo-, as well as deoxyribonucleoside phenylphosphate and enzyme. These conditions are plasmid. Dephosphorylation of DNA and RNA triphosphates (NTPs and dNTPs). CIP is useful in • only used for quantitating enzyme activity. many molecular biology applications such as the **Scale larger reaction volumes proportionally. Supplied in: 50 mM KCl, 10 mM Tris-HCl (pH 8.2 removal of phosphorylated ends of DNA and RNA @ 25°C), 1 mM MgCl , 0.1 mM ZnCl and 50% 3. Purify DNA by gel purification, spin-column or for subsequent use in cloning or end-labeling of 2 2 glycerol. phenol extraction. probes. In cloning, dephosphorylation prevents

Now with CutSmart Reaction Buffer CERTIFICATE OF ANALYSIS Protocol for Dephosphorylation of 5´-ends of DNA 2. CIP is also active in 1X NEBuffers 1.1, 2.1, Physical Purity: Purified to > 95% homogeneity Companion Products Sold Separately: using CIP in Restriction Enzyme Reaction 3.1, as well as NEBuffers 1, 2, 3, 4 and unique as determined by SDS-PAGE analysis using T4 DNA Ligase 1. Digest 1–5 μg of plasmid DNA in a 20 μl reaction as NEBuffer for EcoRI. Coomassie Blue detection. #M0202S 20,000 units follows: 3. CIP activity is enhanced in the presence of #M0202L 100,000 units Heat Inactivation: No #M0202T 20,000 units DNA ≥ 1 µl monovalent salts. 4. CIP is inhibited by metal chelators (e.g. EDTA), #M0202M 100,000 units Restriction Enzyme Buffer (10X) 2 µl References: inorganic phosphate and phosphate analogs. Quick Ligation™ Kit Restriction Endonuclease 1 µl 1. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory #M2200S 30 rxns H O, purified to 20 µl 2 Quality Controls Assays Manual, (2nd ed.), (p. 5.72). Cold Spring Har- #M2200L 150 rxns Exonuclease Activity: Incubation of a 50 µl reaction Note: Scale larger reaction volumes proportionally. bor: Cold Spring Harbor Laboratory Press. Instant Sticky-end Ligase Master Mix containing 50 units of CIP with 1 μg of a mixture 2. Mossner, E., Boll, M. and Pfleiderer, G. (1980) #M0370S 50 rxns 2. Incubate at 37°C for 60 minutes or follow of single and double-stranded [3H] E. coli DNA Hoppe-Seyler's Z. Physiol. Chem. 361, #M0370L 250 rxns manufacturer’s recommendations. for 4 hours at 37°C released < 0.1% of the total 543–549. 3. Add 1 unit of CIP for every 1 pmol of DNA radioactivity. Blunt/TA Ligase Master Mix ends (about 1 μg of a 3 kb plasmid) and incu- #M0367S 50 rxns bate at 37°C for 30–60 minutes. Endonuclease Activity: Incubation of a 50 µl #M0367L 250 rxns 4. Purify DNA by gel purification, spin-column or reaction containing 50 units of CIP with 1 μg of phenol extraction. φX174 RF I DNA for 4 hours at 37°C resulted in < 10% conversion to RF II as determined by 5. Proceed with ligation. ISO 9001 ISO 14001 ISO 13485 agarose gel electrophoresis. Registered Registered Registered Quality Environmental Medical Devices Management Management Usage Notes: RNase Activity: Incubation of a 10 µl reaction New England Biolabs® is a registered trademark of New England Biolabs, Inc. 1. CIP, as are most alkaline phosphatases, is containing 10 units of CIP with 40 ng of fluorescein CUTSMART™ and QUICK LIGATION™ are trademarks of 2+ 2+ a Zn and Mg -dependent enzyme. Our labeled RNA transcript for 4 hours at 37°C resulted New England Biolabs, Inc. 2+ formulation of its storage buffer provides Zn in < 10% degradation of the RNA as determined by 2+ and Mg , which does not require supplemental gel electrophoresis using fluorescence detection. zinc or other additives in reactions with CIP.

Protocol for Dephosphorylation of 5´-ends of DNA 2. CIP is also active in 1X NEBuffers 1.1, 2.1, Physical Purity: Purified to > 95% homogeneity Companion Products Sold Separately: using CIP in Restriction Enzyme Reaction 3.1, as well as NEBuffers 1, 2, 3, 4 and unique as determined by SDS-PAGE analysis using T4 DNA Ligase 1. Digest 1–5 μg of plasmid DNA in a 20 μl reaction as NEBuffer for EcoRI. Coomassie Blue detection. #M0202S 20,000 units follows: 3. CIP activity is enhanced in the presence of #M0202L 100,000 units Heat Inactivation: No #M0202T 20,000 units DNA ≥ 1 µl monovalent salts. 4. CIP is inhibited by metal chelators (e.g. EDTA), #M0202M 100,000 units Restriction Enzyme Buffer (10X) 2 µl References: inorganic phosphate and phosphate analogs. Quick Ligation™ Kit Restriction Endonuclease 1 µl 1. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory #M2200S 30 rxns H O, purified to 20 µl 2 Quality Controls Assays Manual, (2nd ed.), (p. 5.72). Cold Spring Har- #M2200L 150 rxns Exonuclease Activity: Incubation of a 50 µl reaction Note: Scale larger reaction volumes proportionally. bor: Cold Spring Harbor Laboratory Press. Instant Sticky-end Ligase Master Mix containing 50 units of CIP with 1 μg of a mixture 2. Mossner, E., Boll, M. and Pfleiderer, G. (1980) #M0370S 50 rxns 2. Incubate at 37°C for 60 minutes or follow of single and double-stranded [3H] E. coli DNA Hoppe-Seyler's Z. Physiol. Chem. 361, #M0370L 250 rxns manufacturer’s recommendations. for 4 hours at 37°C released < 0.1% of the total 543–549. 3. Add 1 unit of CIP for every 1 pmol of DNA radioactivity. Blunt/TA Ligase Master Mix ends (about 1 μg of a 3 kb plasmid) and incu- #M0367S 50 rxns bate at 37°C for 30–60 minutes. Endonuclease Activity: Incubation of a 50 µl #M0367L 250 rxns 4. Purify DNA by gel purification, spin-column or reaction containing 50 units of CIP with 1 μg of phenol extraction. φX174 RF I DNA for 4 hours at 37°C resulted in < 10% conversion to RF II as determined by 5. Proceed with ligation. ISO 9001 ISO 14001 ISO 13485 agarose gel electrophoresis. Registered Registered Registered Quality Environmental Medical Devices Management Management Usage Notes: RNase Activity: Incubation of a 10 µl reaction New England Biolabs® is a registered trademark of New England Biolabs, Inc. 1. CIP, as are most alkaline phosphatases, is containing 10 units of CIP with 40 ng of fluorescein CUTSMART™ and QUICK LIGATION™ are trademarks of 2+ 2+ a Zn and Mg -dependent enzyme. Our labeled RNA transcript for 4 hours at 37°C resulted New England Biolabs, Inc. 2+ formulation of its storage buffer provides Zn in < 10% degradation of the RNA as determined by 2+ and Mg , which does not require supplemental gel electrophoresis using fluorescence detection. zinc or other additives in reactions with CIP.