Lambda exonuclease, RNase H, RNase HII and uracil-DNA glycosylase were purchased from New England Biolabs and TURBO DNase from Invitrogen and were used at the recommended concentrations and with supplied buffers. RNase A was obtained from Sigma-Aldrich and applied at 100 nM concentration in 0.1 M MES buffer. For each biochip cleavage assay, appropriate nucleic acid substrates were synthesized. In the case of double-stranded substrates, the biochip was synthesized with oligonucleotides capable of self-annealing to form hairpin-loop structures. Cleavage was detected through either the loss of fluorescence of Cy3-terminally labeled sequences, or loss of fluorescence from Cy3-labeled oligonucleotides hybridized to the nuclease substrate. In time course experiments, the enzymatic reaction was stopped by washing the surface followed by drying with argon.
Rnase hii
Manufactured by New England Biolabs
RNase HII is a recombinant DNA endonuclease that cleaves the RNA strand of RNA-DNA hybrids. It is a thermostable enzyme that functions optimally at 37°C.
Automatically generated - may contain errors
Lab products found in correlation
Thermopol buffer, by New England Biolabs (2 mentions)
Uracil dna glycosylase, by New England Biolabs (1 mentions)
Lambda exonuclease, by New England Biolabs (1 mentions)
Rnase h, by New England Biolabs (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Merck Group (1 mentions)
Fla 3000, by Fujifilm (1 mentions)
Taqi v2, by New England Biolabs (1 mentions)
Ecori buffer, by New England Biolabs (1 mentions)
Rcutsmart buffer, by New England Biolabs (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
6 protocols using rnase hii
1
Enzymatic Cleavage of Nucleic Acid Biochips
2
Detecting RNA-DNA Intermediates in Plasmids
3
DNA End Joining Assay with Ku, XRCC4-LigIV, and Pol μ
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The human Ku and XRCC4-Ligase IV complex proteins, as well as the GCG3’ substrate, were prepared as described previously (43 (link)). End joining reactions were carried out in a buffer containing: 25 mM Tris (pH 7.5), 1 mM DTT, 150 mM KCl, 4% glycerol, 50 μg/ml bovine serum albumin, 0.1 mM EDTA and 9% polyethyelene glycol (MW: 8000 kDa). Ligations were carried out by adding (final concentrations) 10 nM Ku, 20 nM XRCC4-LigaseIV complex and 0.5 nM Pol μ, to a mixture containing 2 nM DNA substrate, 2 mM Mg2+, 100 ng supercoiled DNA and 100 μM CTP or dCTP. The reactions were carried out at 37°C for 10 min, stopped by addition of EDTA and SDS, and either purified by a Minelute cartridge (Qiagen) or extracted once with a 1:1 mixture of phenol and chloroform. RNase HII (New England Biolabs) digestion of joined products was carried out overnight as per the manufacturer's instructions. Ligation products were resolved using a 5% native PAGE gel and visualized using a Typhoon Imager for qualitative comparisons. End joining products were quantified using qPCR, with primers specific for substrate and product.
4
RNase HI and RNase HII Detection
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Treatments of 0.5 μg of the total genomic DNA with RNase HI (Takara) or RNaseHII (NEB) were performed as described (18 (link)). It was critical to run the reaction samples on the gel followed up by electric transfer to separate RNase HI from the substrate. The enzyme tightly binds to the substrate (even at 0°C in water) and completely blocks immunodetection if a reaction mix is directly used in the dot-blot procedure.
5
Enzymatic DNA Cleavage Protocols
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EcoRI (New England BioLabs Inc. Cat#R0101S) activity in perchlorate solutions generally consisted of 0.05 U/µL enzyme, 1X EcoRI buffer (New England BioLabs Inc. Cat#B7006S), and 1 µM duplex DNA (see Supplementary Table 1 ). NaX concentration varied per experiment as noted in the text. Reactions were started by incubating at 37 °C. Reactions were stopped and cleaned up with the addition of 75% ethanol. DNA cleavage was quantified using a 5′-fluorescein label and a 20% urea-PAGE gel.
RNase HII (New England BioLabs Inc. Cat#M0288S) was assessed with the same protocol but in 1X ThermoPol Buffer (New England BioLabs Inc. Cat#B9004S).
TaqI-v2 ((New England BioLabs Inc. Cat#R0149S) was assessed with the same protocol but in 1X rCutSmart Buffer (New England BioLabs Inc. Cat#B6004S) and incubated at 65 °C. DNA was resolved on a 10% urea-PAGE gel.
All gels were analyzed using an Omega Lum G Imaging System using the Omega Lum Image Capture Software V 2.1.2017.0. GelQuant (GelQuant.NET V 1.8.2) was used for quantification.
RNase HII (New England BioLabs Inc. Cat#M0288S) was assessed with the same protocol but in 1X ThermoPol Buffer (New England BioLabs Inc. Cat#B9004S).
TaqI-v2 ((New England BioLabs Inc. Cat#R0149S) was assessed with the same protocol but in 1X rCutSmart Buffer (New England BioLabs Inc. Cat#B6004S) and incubated at 65 °C. DNA was resolved on a 10% urea-PAGE gel.
All gels were analyzed using an Omega Lum G Imaging System using the Omega Lum Image Capture Software V 2.1.2017.0. GelQuant (GelQuant.NET V 1.8.2) was used for quantification.
6
Alkaline Gel Electrophoresis of Genomic DNA
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Rnaseh2b +/+ ;Tp53 -/-, Rnaseh2b +/-;Tp53 -/-, and Rnaseh2b -/-;Tp53 -/-MEFs [56] were transiently transfected with Rnaseh2b constructs or empty vector and cultured for 48h. MEFs were mechanically disrupted in ice-cold lysis buffer (20 mM Tris-HCl, pH 7.5, 75 mM NaCl, 50 mM EDTA), incubated with 100 µg/ml proteinase K, and completely lysed using sodium dodecyl sulphate at a final concentration of 1%. Nucleic acids were sequentially extracted with TEequilibrated phenol, phenol:chloroform:isoamylalcohol (25:24:1), and chloroform, precipitated with isopropanol, washed with 75% ethanol, and dissolved in water over night at 4°C. Digestion with RNase HII (NEB, Frankfurt am Main, Germany) was carried out for 1h at 37°C, and nucleic acids were ethanol precipitated and dissolved in water. For alkaline gel electrophoresis [48] , one volume of loading buffer (300 mM NaOH, 6 mM EDTA, 7.5% Ficoll (Type 400), 0.35% Orange G)
was added to each sample, and DNA was separated on a 0.5% agarose gel (50 mM NaCl, 1mM EDTA) using an alkaline running buffer (50 mM NaOH, 1 mM NaCl) for 16h at 16V (B1 mini-gel format; Thermo Fisher Scientific). After electrophoresis, gels were neutralized with 0.7 M Tris/HCl buffer (pH 8.0) containing 1.5 M NaCl for 1h at RT and stained with SYBR Gold for 1h at RT
(1:10,000 dilution; Thermo Fisher Scientific).
was added to each sample, and DNA was separated on a 0.5% agarose gel (50 mM NaCl, 1mM EDTA) using an alkaline running buffer (50 mM NaOH, 1 mM NaCl) for 16h at 16V (B1 mini-gel format; Thermo Fisher Scientific). After electrophoresis, gels were neutralized with 0.7 M Tris/HCl buffer (pH 8.0) containing 1.5 M NaCl for 1h at RT and stained with SYBR Gold for 1h at RT
(1:10,000 dilution; Thermo Fisher Scientific).
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