An endoribonuclease that degrades the RNA portion of DNA:RNA hybrids
E. coli strain containing an overproducing clone of E. coli RNase H.
E. coli RNase H is an endoribonuclease that degrades the RNA portion of DNA:RNA hybrids(1). It is a key enzyme in the Okayama-Berg and Gubler-Hoffman cDNA synthesis and cloning procedures, in which it is employed to remove the mRNA during second-strand cDNA synthesis(2,3). RNase H is useful for the removal of poly(A) tails on mRNAs after hybridization with oligo (dT)(4 - 6). It has also been used for the specific fragmentation of RNA after hybridization of the RNA with oligodeoxynucleotides(7).
Activators: Mg2+ or Mn2+
Molecular Weight: 21 kDa
Tested for contaminating non-specific endonucleases, exonucleases and ribonucleases.
20mM Tris-HCl (pH 7.9), 100mM KCl, 10mM MgCl2, 0.1mM EDTA, 0.1mM DTT, 50% glycerol.
Reaction (100 μl) contains 20mM Tris HCl (pH 8.0), 100mM KCl, 10mM MgCl2, 1mM DTT, 0.5 nmol poly(rA):poly(dT), enzyme.
One unit catalyzes the hydrolysis of 1 nmol of RNA in radiolabeled poly(A)-poly(dT) to acid-soluble material in20 min at 37°C.
PROTOCOL TO REMOVE mRNA DURING SECOND STRAND cDNA SYNTHESIS
- Prepare 10X Second Strand Synthesis Buffer: 188mM Tris-HCl (pH 8.3), 900mM KCl, 46mM MgCl2, 37.5mM DTT.
- To the first strand reaction mixture, add the following components in the order indicated. Maintain all components on ice during the additions:
Sterile (DEPC-treated) water 233 μl
10mM dNTP mix (10mM each) 6 μl
10X Second Strand Synthesis Buffer 32 μl
[α-32P]dCTP (400-3000 Ci/mmol) 1 μl
E. coli DNA Polymerase I (15 units/μl) 6 μl
Note: These volumes are based on a 40 μl first-strand reaction mix volume.
- Mix the contents of the tube briefly and incubate the reaction for 2 hrs at 16°C. Do not let the temperature rise above 16°C.
- Place the tube on ice and add 25 μl of 250mM EDTA.
- Remove 10 μl from the reaction mixture and add 90 μl of water. Spot 5 μl onto a glass fiber filter and let dry. This will be used to determine the amount of 32P in the reaction mixture.
- Remove a second 10 μl aliquot from the reaction mixture and spot onto another glass fiber filter. Wash the filter with TCA and ethanol. This will be used to determine the incorporation of 32P into the second strand cDNA. Count the filters from steps 5 and 6.
- Extract the remainder of the reaction with phenol:chloroform:isoamyl alcohol and precipitate with ammonium acetate and ethanol.
- Dissolve the pellet in 200 μl of sterile TE buffer and precipitate a second time with ammonium acetate and ethanol.
- Dissolve the dry pellet in 20 μl of sterile TE buffer. Be sure to thoroughly resuspend the pellet. It is advisable to vortex and centrifuge alternately several times to assure full recovery of the material from the pellet. Remove 2 μl for later gel analysis if desired.
- To the remaining 18 μl add the following in the order indicated:
5X RNase H Buffer 20 μl
Sterile (DEPC-Treated) water 61 μl
RNase H 5 units
- Vortex the tube, transfer to a 37°C water bath and incubate for 20 min.
- Place the tube on ice and add 2mM EDTA to stop the reaction.
- Extract the reaction with phenol:chloroform:isoamyl alcohol and precipitate with ammonium acetate and ethanol.
- Dissolve the pellet in 200 μl of TE buffer and reprecipitate with ammonium acetate and ethanol.
- Dissolve the pellet thoroughly in 20 μl of TE buffer and count 1 μl by TCA precipitation on a glass fiber filter to determine the recovery of double-stranded cDNA. The remainder may be used for gel analysis and cloning by tailing.
5X RNase H Reaction Buffer (1 ml included, PN 71530):
100mM Tris-HCl (pH 7.5), 100mM KCl, 50mM MgCl2, 0.5mM EDTA, 0.5mM DTT
- CROUCH, R. J. (1981) in Gene Amplification And Analysis, eds. J. G. Chirikjian and
T. S. Papas, (Elsevier/North Holland, Inc., New York) 2, 217-228.
- OKAYAMA, H. AND BERG, P. (1982) Mol. Cell Biol. 2, 161-170.
- GUBLER, U. AND HOFFMAN, B. J. (1983) Gene 25, 263-269.
- VOURNAKIS, J. N., EFSTRATIADIS, A. AND KAFATOS, F. C. (1975) Proc. Natl. Acad. Sci. USA 72, 2959-2963.
- NARAYAN, P., AYERS, D. F., ROTTMAN, F. M., MARONEY, P. A. AND NILSEN,
T. W. (1987) Mol. Cell. Biol. 7,1572-1575.
- DAVIS, R. E., DAVIS, A. H., CARROLL, S. M., RAJKOVIC, A. AND ROTTMAN, F. M.(1988) Mol. Cell Biol. 8, 4745-4755.
- DONIS-KELLER, H. (1979) Nucl. Acids Res. 7,179-192.
- GOODWIN, E. C. AND ROTTMAN, F. M. (1992) Nucl. Acids Res. 20, 916.
- Removes the mRNA strand of the RNA:DNA duplex during second strand cDNA synthesis.
- Removal of poly (A) tails on mRNA after hybridization with oligo dT.
- Making specific fragmentation of RNA after hybridization of RNA with oligodeoxynucleotides.
- Site-specific cleavage of RNA(7).
- Study of in vitro polyadenylation reaction products(8).
Shipped on dry ice. Store at -20°C.
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