Ultrahyb

Manufactured by Thermo Fisher Scientific

Sourced in United States

ULTRAhyb is a prehybridization/hybridization buffer designed for use in Northern and Southern hybridization applications. It is a ready-to-use solution that helps to improve the sensitivity and specificity of nucleic acid hybridization experiments.

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Lab products found in correlation

60 protocols using ultrahyb

Mouse Retina cDNA Preparation and vps26A Analysis

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Mouse retina cDNA was prepared as described [135] (link). vps26A was PCR-amplified using primers 5′-GAGTTTTCTTGGAGGCTTTTTTGGTCC-3′ and 5′-TTACATCTCAGGCTGCTCCGCAGAGG-3′ and a 30 ng cDNA template. The PCR product was cloned into pBluescript by blunt-end ligation and verified by sequencing. DNA probes were generated from gel-purified insert (excised with EcoRI and HindIII) by random-prime labeling with [α-³²P]dCTP using the DECAprimeII kit (Ambion). Unincorporated nucleotides were removed with a MicroBio-Spin 30 column (Bio-Rad).
For Northern blot analysis, total RNA was prepared from retinas of 3–4-mo-old CD-1 mice by homogenization in TRI reagent (Ambion) using a motorized pestle and passage through a 20-gauge needle, extraction with 1-bromo-3-chloro-propane, and purification with the RNeasy kit (Qiagen). We resolved 10 µg of total RNA on a formaldehyde-agarose gel and transferred it to a Hybond-N+ nylon membrane (GE Healthcare) by capillary transfer. Membranes were prehybridized in ULTRAhyb (Ambion) for 1 h, then incubated at 42°C overnight with a ∼2.5×10⁷ cpm denatured probe diluted in ULTRAhyb. Blots were washed several times in 2× SSC+0.1% SDS and several times in 0.1× SSC+0.1% SDS. Washes were performed at 42–45°C. For imaging, blots were exposed to a storage phosphor screen and scanned on a Typhoon (GE Healthcare).

Wang S., Tan K.L., Agosto M.A., Xiong B., Yamamoto S., Sandoval H., Jaiswal M., Bayat V., Zhang K., Charng W.L., David G., Duraine L., Venkatachalam K., Wensel T.G, & Bellen H.J. (2014). The Retromer Complex Is Required for Rhodopsin Recycling and Its Loss Leads to Photoreceptor Degeneration. PLoS Biology, 12(4), e1001847.

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Isolation and Detection of miR-122

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RNA was isolated using TRIzol reagent (Ambion) according to the manufacturer’s instructions. Total RNA from human liver was purchased from Agilent Technologies (Cat. No. 540017). Northern blotting was carried out using standard procedures on equal molar quantities of RNA. Membranes were probed with a random-primed ³²P-labelled DNA fragment corresponding to nt 3077-3707 (exon probe) and nt 1563-2005 (intron probe) of pri-miR-122 in Ultrahyb (Ambion). A fragment corresponding to nt 685-1171 of γ-actin was used as a loading control. For miRNA northern blots, the small RNA fraction was isolated by dissolving total RNA in 300μL of TE buffer with addition of equal amounts of PEG solution (20% PEG 8000, 2M NaCl). Samples were mixed and incubated for at least 30 min on ice, followed by centrifugation at 14,000g for 15min and isopropanol precipitation of the supernatant. Small RNA were run on a 18% polyacrylamide (19:1) urea gel and analyzed as described before⁵⁴ using a ³²P-end-labeled oligonucleotide complementary to miR-122.

Dhir A., Dhir S., Proudfoot N.J, & Jopling C.L. (2015). Microprocessor mediates transcriptional termination in long noncoding microRNA genes. Nature structural & molecular biology, 22(4), 319-327.

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Isolation and Detection of miR-122

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Dhir A., Dhir S., Proudfoot N.J, & Jopling C.L. (2015). Microprocessor mediates transcriptional termination in long noncoding microRNA genes. Nature structural & molecular biology, 22(4), 319-327.

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Strand-specific Southern Blot Analysis

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Strand-specific Southern blot was performed as described^{6 (link)}. Briefly, AflIII and AseI digested samples were separated on a 7% polyacrylamide gel and transferred to a nylone membrane (Hybond-N+, Amersham). After transfer, the membrane was rinsed in 4X SSC for 5 minutes, and UV irradiated to crosslink the DNA to the membrane. The membrane was then pre-hybridized with 25 ml hybridization buffer (ULTRAhyb from Ambion) for at least 3 hours at 42 °C. Strand-specific probes generated by a PCR based primer extension reaction^{6 (link)}, were added to the hybridization buffer and incubated with the membrane at 42 °C overnight. After overnight hybridization, the membrane was washed 2 times with 2X SSC, 0.1% SDS for 5 minutes at 42 °C. The dried membrane was exposed to a phosphorimager screen.
Uncropped images of gels and autoradiographs used in this study can be found in Supplementary Data Set 1.

Zhang J., Dewar J.M., Budzowska M., Motnenko A., Cohn M.A, & Walter J.C. (2015). DNA interstrand cross-link repair requires replication fork convergence. Nature structural & molecular biology, 22(3), 242-247.

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Northern Blot Analysis of Lung RNA

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Northern blots were performed as previously described with some modifications^{1 (link),49 (link)}. Briefly, total RNA extracted from lungs was heat-denatured for 15 min at 75 °C before being electrophoresed on a 1.2% agarose gel containing 7% formaldehyde, alongside an RNA ladder (Life Technologies). Gels were run overnight at 4 °C in MOPS buffer (20 mM MOPS, 8 mM sodium acetate, 1 mM EDTA, pH 7.0). RNA was transferred by capillary transfer to a nylon membrane (Hybond N+, Amersham Biosciences) overnight after which the membrane was crosslinked with UV light (120 mJ/cm²) and hybridised with [α³²P]-dCTP and [α³²P]-dATP labelled probes diluted in hybridisation buffer (Ambion Ultrahyb) overnight at 42 °C. Primer sequences used to generate the probes are listed in Table 1.

Rigby R.E., Wise H.M., Smith N., Digard P, & Rehwinkel J. (2019). PA-X antagonises MAVS-dependent accumulation of early type I interferon messenger RNAs during influenza A virus infection. Scientific Reports, 9, 7216.

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RNA Analysis via Gel Electrophoresis

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For polyacrylamide gels, 3 μg of each RNA sample was prepared with formamide loading buffer and loaded on 10% polyacrylamide Tris-borate-EDTA (TBE)-urea gels and electrophoresed at 60–85 V unless noted otherwise. The RNA was then transferred to a Zeta-Probe GT membrane (Bio-Rad) using a Trans-Blot SD semidry transfer apparatus (Bio-rad) following the manufacturer's guidelines. For agarose gels, 10 μg of each sample was prepared with formaldehyde and formamide loading buffer and loaded on 1.2% agarose 3-(N-morpholino)propanesulfonic (MOPS) gels. Gels were electrophoresed at 65 V and transferred to Zeta-Probe GT membrane by overnight capillary transfer. After transfer, membranes were UV-crosslinked and hybridized overnight with 100 ng/ml of 5′ biotinylated DNA probes (Supplementary Table S2) in ULTRAhyb (Ambion) hybridization buffer at 42°C. Blots were developed using the BrightStar BioDetect kit protocol (Ambion).

Cameron T.A., Matz L.M., Sinha D, & De Lay N.R. (2019). Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments. Nucleic Acids Research, 47(16), 8821-8837.

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HCV and Rab27a Expression Analysis

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Huh7 cells were washed once with PBS and total RNA was extracted using TRIzol (Invitrogen) following the manufacturer’s protocol. Ten μg of total RNA in RNA loading buffer (32% formamide, 1x MOPS-EDTA-Sodium acetate (MESA, Sigma) and 4.4% formaldehyde) was denatured at 65°C for 10 min and separated in a 1% agarose gel containing 1x MESA and 3.7% formaldehyde. The RNA was transferred and UV crosslinked to a Zeta-probe membrane (Bio-Rad). The membrane was hybridized using the ExpressHyb hybridization buffer (Clontech) or ULTRAhyb (Ambion) and α-³²P dATP-RadPrime DNA labelled probes (Invitrogen) complementary to HCV (nucleotides 84–374), Rab27a (nucleotides 664–1145), or actin (nucleotides 685–1171). Autoradiographs were quantified using ImageQuant (GE Healthcare).

Chen T.C., Hsieh C.H, & Sarnow P. (2015). Supporting Role for GTPase Rab27a in Hepatitis C Virus RNA Replication through a Novel miR-122-Mediated Effect. PLoS Pathogens, 11(8), e1005116.

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Protein and RNA extraction and analysis

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Cells were lysed in NP40 buffer (25 mM HEPES-KOH, 150 mM KCl, 1.5 mM MgCl2, 0.5% NP40, 10% Glycerol, pH 7.5) supplemented with protease inhibitors (Roche, 11836153001) for 20 min on ice. Lysate was clarified by centrifugation at 16,000 xg for 10 min and quantified by BCA assay (Pierce, 23225). For Western blotting, 10-50 μg was separated by SDS-PAGE, transferred onto PVDF membrane (GE Healthcare, RPN303F), and blotted according to standard protocols. 5% milk in TBST (0.1% Tween) was used for all blocking and antibody incubation steps; TBST (0.1% Tween) was used for all washes. For Northern blotting, RNA was extracted with TRIzol (Life Technologies, 15596-026), separated using 5% urea TBE gel electrophoresis, transferred onto Hybond-N membrane (GE Healthcare, RPN303N), and hybridized with ULTRAhyb (Ambion, AM8670). TERC probe was generated by random body labeling with full length TERC cDNA.

Freund A., Zhong F.L., Venteicher A.S., Meng Z., Veenstra T.D., Frydman J, & Artandi S.E. (2014). Proteostatic control of telomerase function through TRiC-mediated folding of TCAB1. Cell, 159(6), 1389-1403.

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Quantitative Analysis of Small RNAs

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Two micrograms of each RNA sample was loaded on 5% or 10% Criterion TBE-urea precast gels (Bio-Rad) and electrophoresed at 70 V. Next, the RNA samples were transferred to a Zeta-Probe GT membrane (Bio-Rad) using a Trans-Blot SD semidry transfer apparatus (Bio-rad) following manufacturer's guidelines. Transferred RNA was UV crosslinked and hybridized overnight with 100 ng/mL of 5′ biotinylated DNA probe (Supplemental Table S2) in ULTRAhyb (Ambion) hybridization buffer at 42°C. Blots were developed using a BrightStar BioDetect kit protocol (Ambion), imaged with a ChemiDoc MP imager (Bio-Rad) and quantified using Image Lab software version 5.2.1 (Bio-Rad). Signal intensity corresponding to each sRNA or mRNA was normalized to that of either ssrA or 5S rRNA, which served as internal loading controls. Decay curves corresponding to RNA stability time course experiments were generated by using GraphPad Prism version 5.0.

Sinha D., Matz L.M., Cameron T.A, & De Lay N.R. (2018). Poly(A) polymerase is required for RyhB sRNA stability and function in Escherichia coli. RNA, 24(11), 1496-1511.

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Northern Blotting of Total RNA

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Total RNA (10 μg) was separated on a 8% polyacrylamide-7M urea gel in 1X TBE (90 mM Tris-borate 2 mM EDTA). The RNA was transferred to a Zeta-Probe GT blotting membrane (Bio-Rad) at 20V for ∼16 h at 4°C in 0.5X TBE. After transfer, membranes were allowed to dry, UV cross-linked on both sides, and incubated overnight at 45°C in UltraHyb (Ambion) hybridization buffer and oligonucleotides 5′-end-labeled with ³²P-ATP with T4 polynucleotide kinase (New England Biolabs). Subsequently, membranes were washed once with 2X SSC (150 mM NaCl 15 mM sodium citrate) 0.1%SDS, incubated 10 min at 45°C in 2X SSC 0.1%SDS, and washed 5X with 0.2X SSC 0.1% SDS. After washing, air-dried membranes were exposed to HyBlot CL film (Denville Scientific) at −80°C.

Hao Y., Updegrove T.B., Livingston N.N, & Storz G. (2016). Protection against deleterious nitrogen compounds: role of σS-dependent small RNAs encoded adjacent to sdiA. Nucleic Acids Research, 44(14), 6935-6948.

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