Ribonuclease H, E. coli

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Reagents Supplied

10X E. coli Ribonuclease H Reaction Buffer

Applications

• Hybridization of a synthetic DNA oligomer to a complementary single-stranded region of a RNA molecule can be used to create a site that can be cleaved by RNase H (1)
• Used to remove RNA strand before second strand cDNA synthesis (2, 3)
• Detects RNA-DNA regions in naturally occurring double-stranded DNA (4)
• Used to analyze in vitro polyadenylation reaction products (5)

Unit Definition

One unit is the amount of enzyme required to produce 1 nmol of acid soluble ribonucleotide from [32P]poly(A).poly(dT) at 37°C in 20 minutes

Reagents Supplied

10X Ribonuclease H Reaction Buffer

Heat Inactivation

65ºC for 15 minutes

Concentration

1 – 5 units/µl

Assay Conditions

37 mM Tri-HCl (pH 8.0)
14.8 mM KCl
7.4 mM MgCl₂
2.2 mM β-mercaptoethanol
0.6 nmoles [32P]poly(A)-poly(dT)
Reaction volume of 25μl (6)

Storage Buffer

20 mM Tris-HCl (pH 7.5) 
300 mM KCl
1.0 mM DTT
7 mM EDTA
20 mM magnesium acetate
50% (v/v) glycerol

Storage Conditions

Store at -20°C
Product shipped on Dry Ice

References

(1) Donis-Keller, H. (1979) Nucleic Acids Res. 7, 179-182
(2) Okayama, H. and Berg, P. (1982) Mol. Cell Bio. 2, 161-170
(3) Gubler, U. and Hoffman, B.J. (1983) Gene 25, 263-269
(4) Keller, W. and Crouch, R. (1972) Proc. Natl. Acad. Sci. USA 69, 3360-3364
(5) Goodwin, E.C. and Rottman, F.M. (1982) Nucl. Acids Res. 20, 916
(6) Hillenbrand, G. and Staudenbauer, W.L. (1982) Nucl. Acids Res. 10, 833-852