HK™ Thermolabile Phosphatase

Catalogue number

Pack size

H92100

1U/µl 100 MBU

Derived from an Antarctic bacterium, HK™ (Heat-Killable) Thermolabile Phosphatase is free of DNase and RNase activities and can be easily heat-killed. The enzyme removes phosphates from protruding 5'-ends of double-stranded DNA generated by many restriction enzymes, but is not active on recessed 5'-ends and has low activity on blunt ends. HK™ Phosphatase is as effective as calf intestinal phosphatase (CIP) and bacterial alkaline phosphatase (BAP) in dephosphorylating RNA. However, unlike CIP and BAP, this patented* enzyme is completely and irreversibly inactivated by incubation at 65°C or higher for 15 minutes.

HK™ Phosphatase greatly simplifies the dephosphorylation of DNA vectors prior to cloning, preventing vector religation. Because HK™ Phosphatase is active in many enzyme buffers, all steps leading to cloning can be performed in a single tube without organic extractions.

Table 1. Dephosphorylation of RNA by HK Phosphatase,BAP, and CIP

Phosphatase	% of RNA Dephosphorylated
HK Phosphatase	93
BAP	95
CIP	96
Each phosphatase was incubated for 1 hour with 1µg of 5´- 32P-labelled RNA under the recommended reaction conditions. Dephosphorylation of the RNA was determined by measurement of acid-soluble radioactivity.

Table 2. Phosphatase Activity Remaining After Heat Treatment at 70°C for 15 Minutes

Phosphatase	% of RNA Dephosphorylated
HK Phosphatase	0
BAP	41
CIP	62
Each phosphatase was incubated at 70°C for 15 minutes. Remaining phosphatase activity was then determined by incubation with 5´- 32P-labelled RNA as in Table 1.

Unit Definition:

One Molecular Biology Unit (MBU) dephosphorylates 1µg of Hind III-digested pUC19 DNA in 1 hour at 30°C in 33mM Tris-acetate, pH 7.8, 66mM potassium acetate, 10mM magnesium acetate, 0.5mM DTT, and 5 mM CaCl2.

Storage Buffer:

50% glycerol containing 25mM Tris-HCl, pH 7.5, 0.1M NaCl, 0.1mM EDTA, 5mM CaCl2, and 0.01% Triton® X-100.

HK Phosphatase 10X Reaction Buffer:

330mM Tris-acetate, pH 7.8, 660mM potassium acetate, 100mM magnesium acetate, and 5mM DTT. CaCl2 is provided as a separate solution and should be added to a final concentration of 5mM.

Quality Control:

Every lot of HK™ Phosphatase is functionally tested and required to meet our Cloning Quality Standard: greater than 90% of transformant colonies must contain recombinant plasmids using HK™ Phosphatase-treated BamHI-cut pBR322 as the cloning vector. HK Phosphatase is free of detectable RNase and exo- and endonuclease activities.

Dephosphorylation of vector DNA with HK™ Phosphatase reduces the frequency of non-recombinant ('background') transformants. E. coli cells were transformed with 300ng of either untreated (A) or HK Phosphatase-treated (B) BamH I-digested pBR322 that had been incubated with T4 DNA Ligase and 7.5ng of a 208-bp BamH I fragment containing the lac operator. Colonies containing recombinant plasmids are blue (dark colonies in photos) when plated on medium containing X-gal because the lac operator titrates the lac repressor, allowing expression of host chromosomal ß-galactosidase. Note: the near-total absence of non-recombinant transformants (smaller, lighter colonies) in the cells transformed with HK Phosphatase-treated DNA (B). APex™ Heat-Labile Alkaline Phosphatase removes 5'-phosphates from both recessed and blunt ends, in addition to being more active than HK Phosphatase in removing phosphates from 5'-overhang ends.

Last modified: 2008-05-14 16:46:46

Applications

• Dephosphorylation of DNA vectors prior to cloning
• Dephosphorylation of RNA

Benefits

• Completely and irreversibly heat-inactivated at 65°C
• Active in most enzyme buffers - perform several cloning steps (restriction enzyme digestion, DNA phosphorylation, enzyme inactivation, DNA ligation) in a single tube without organic extractions or ethanol precipitation. Following ligation, add DNA directly to competent cells for transformation

Last modified: 2008-05-14 16:46:46

Protocols for: HK™ Thermolabile Phosphatase

HK™ Thermolabile Phosphatase Protocol

(catalogue number H92025 / H92050 / H92100)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46

References

1. Hoffman, L. and Jendrisak, J. (1990) Gene 88, 97.
2. Kobori, J.A. et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6691..

Last modified: 2008-05-14 16:46:46