α p32 utp

Manufactured by PerkinElmer

α-P32-UTP is a radioactive ribonucleotide triphosphate labeled with the phosphorus-32 (P32) isotope. It is a core component used in various molecular biology and biochemical applications, such as nucleic acid labeling and detection.

Automatically generated - may contain errors

Lab products found in correlation

5 protocols using α p32 utp

Extraction and Analysis of Small RNAs

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total RNA was extracted from mid-log phase cells grown at 18°C for 3 days using the hot phenol method in equal volumes of AES buffer (50mM sodium acetate pH 5.3, 10mM DTA, 1% SDS) and acid-phenol. The mixture was incubated at 65°C and vortexed every minute for 5 minutes. Afterward, the slurry was transferred to Maxtract High Density tubes (QIAGEN) and RNA was purified by chloroform extraction. Purified RNA was precipitated using Glycoblue (Thermo Fisher Scientific), sodium acetate pH 5.3 and isopropanol. 5μg of total RNA per sample was loaded per lane in a 1% formaldehyde agarose gel. T7 MAXIscript kit (Ambion) was used to generate α-P³²-UTP (PerkinElmer) labeled RNA probes and hybridizations were carried out using the NorthernMax kit (Ambion).
For probing centromeric small interfering RNAs (siRNAs), siRNAs were purified from exponentially growing cells with mirVana miRNA isolation kit (Thermo Fisher Scientific). 20μg of small RNAs (< 200nt) was resolved on 15% urea-PAGE and electro-transferred to HybondTM-N+ (Thermo Fisher Scientific) membrane in 0.5xTBE for 1 hour at 100V. After UV crosslinking the membrane was probed for siRNAs by hybridization with α-P³²-UTP (PerkinElmer) labeled RNA probes (~50nt) corresponding to dg/dh sequence in UltraHyb-oligo hybridization buffer (Thermo Fisher Scientific).

Vo T.V., Dhakshnamoorthy J., Larkin M., Zofall M., Thillainadesan G., Balachandran V., Holla S., Wheeler D, & Grewal S.I. (2019). CPF Recruitment to Non-canonical Transcription Termination Sites Triggers Heterochromatin Assembly and Gene Silencing. Cell reports, 28(1), 267-281.e5.

+ Open protocol

+ Expand

Centromeric siRNA Northern Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Centromeric small interfering RNAs (siRNAs) northern blot analysis was performed as described previously (Sugiyama et al., 2007 (link)). Small RNAs (< 200 nt) were purified from mid-log phase cells with mirVana miRNA isolation kit (Thermo Fisher Scientific). Twenty μg of small RNAs were resolved on a 15% denaturing acrylamide gel and transferred to HybondTM-N+(Thermo Fisher Scientific) membrane in 0.5x TBE for 1 h at 100V. After UV crosslinking, the membrane was hybridized with α-P³²-UTP (PerkinElmer) labeled RNA probes (~50nt) corresponding to dg sequence in UltraHyb-oligo hybridization buffer (Thermo Fisher Scientific).

Holla S., Dhakshnamoorthy J., Folco H.D., Balachandran V., Xiao H., Sun L.L., Wheeler D., Zofall M, & Grewal S.I. (2019). Positioning heterochromatin at the nuclear periphery suppresses histone turnover to promote epigenetic inheritance. Cell, 180(1), 150-164.e15.

+ Open protocol

+ Expand

In Vitro Transcription Assay for NasR Regulation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Each 10 μl of reaction mixture included 1× E. coli RNA Polymerase Reaction Buffer (New England Biolabs), 500 μM ATP, 500 μM CTP, 500 μM GTP, 500 μM UTP, 250 μM DNA template (JRG105/MG327 PCR product from pMG1127), 8 μM purified NasR, 0.5 units of E. coli RNAP, ~3 pmol of α‐P³²‐UTP (Perkin Elmer). The 10 μl of reaction mixture was then incubated at 37°C for 1 hr and then one volume of 2× denaturing gel loading buffer (0.09 M tris, 0.09 M borate, 10 mM EDTA (pH 8.0), 18 M urea, 20% sucrose, 0.1% SDS, 0.05% bromophenol blue, 0.05% xylene cyanol) was added. The resulting mixture was resolved by urea‐denaturing PAGE on a 8% gel. The resulting gel was then dried and exposed to a phosphor screen for analysis on a phosphor imager. Band intensities were quantified using the FIJI/ImageJ gel quantification plugin (Schindelin et al., 2012; Schneider et al., 2012).

Goodson J.R., Zhang C., Trettel D., Ailinger H.E., Lee P.E., Spirito C.M, & Winkler W.C. (2020). An autoinhibitory mechanism controls RNA‐binding activity of the nitrate‐sensing protein NasR. Molecular Microbiology, 114(2), 348-360.

+ Open protocol

+ Expand

In Vitro Transcription Assay for NasR

Check if the same lab product or an alternative is used in the 5 most similar protocols

Each 10 μL reaction mixture included 1x E. coli RNA Polymerase Reaction Buffer (New England Biolabs), 500 μM ATP, 500 μM CTP, 500 μM GTP, 500 μM UTP, 250 μM DNA template (JRG105/MG327 PCR product from pMG1127), 8 μM purified NasR, 0.5 units of E. coli RNAP, ~3 pmol of α-P³²-UTP (Perkin Elmer). The 10 μL reaction mixture was then incubated at 37 °C for one hour and then one volume of 2x denaturing gel loading buffer (0.09 M tris, 0.09 M borate, 10 mM EDTA (pH 8.0), 18 M urea, 20% sucrose, 0.1% SDS, 0.05% bromophenol blue, 0.05% xylene cyanol) was added. The resulting mixture was resolved by urea-denaturing PAGE on a 8% gel. The resulting gel was then dried and exposed to a phosphor screen for analysis on a phosphor imager. Band intensities were quantified using the FIJI/ImageJ gel quantification plugin(Schindelin et al., 2012 (link); Schneider et al., 2012 (link)).

+ Open protocol

+ Expand

Radioactive RNA Transcription and Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Plasmids were linearized by EcoR1 restriction and 100 ng were transcribed using T7 transcription kit (Ambion) in presence of 1 µl of [αP³²]-UTP (Perkin Elmer), analyzed on 8% denaturing polyacrylamide and quantified with LS-6500 counter (Beckman). Non-labeled transcripts were synthesized using the Megascript T7 kit (Ambion). After transcription, 1 unit of DNase I (Invitrogen, Carlsbad, CA) was added, and the sample was incubated for additional 30 min at 37 °C. Transcribed RNAs were then purified by micro Bio-Spin 6 chromatography columns (Bio-rad) according to the manufacturer’s instructions. The sizes of RNAs were checked by gel electrophoresis on a denaturing 6% polyacrylamide gel.

Sellier C., Cerro-Herreros E., Blatter M., Freyermuth F., Gaucherot A., Ruffenach F., Sarkar P., Puymirat J., Udd B., Day J.W., Meola G., Bassez G., Fujimura H., Takahashi M.P., Schoser B., Furling D., Artero R., Allain F.H., Llamusi B, & Charlet-Berguerand N. (2018). rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences. Nature Communications, 9, 2009.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!