Decalabel dna labelling kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The DecaLabel DNA Labelling Kit is a tool designed for the labeling of DNA samples. The kit provides the necessary reagents and components to facilitate the incorporation of fluorescent or other types of labels into DNA molecules, enabling their detection and analysis in various downstream applications.

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Lab products found in correlation

Amersham hybond nx membrane, by GE Healthcare (3 mentions) Nick column, by GE Healthcare (1 mentions) α 32p dctp, by PerkinElmer (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Hybond xl, by GE Healthcare (1 mentions) Imagequant, by GE Healthcare (1 mentions) Phosphoimager fla 7000, by GE Healthcare (1 mentions) Tru1i restriction endonuclease, by Thermo Fisher Scientific (1 mentions) Phosphorimager screen, by Cytiva (1 mentions) Hybond nx membrane, by GE Healthcare (1 mentions) Hybond xl membrane, by GE Healthcare (1 mentions) Fla7000 imager, by Fujifilm (1 mentions)

9 protocols using decalabel dna labelling kit

Quantitative Northern Blot Analysis of tRNA and 5S rRNA

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RNA was prepared in Trizol (Invitrogen) according to the manufacturers recommendations, resuspended in formamide and 1 μg separated on a 15% polyacrylamide/TBE gel. RNA was then transferred by wet blotting onto Immobilon NY+ nitrocellulose membranes (Millipore, Watford, UK) and crosslinked in a Stratalinker at 120 mJoules. The crosslinked membranes were transferred to hybridisation tubes and pre-hybridised for an hour at 42°C in hybridisation solution (50% formamide, 10% dextran sulfate, 1% SDS, 5x Denhardt's solution in 5x SSPE buffer). Random-labelled probes were generated by incubating 100 ng PCR amplified tRNA and 5S rRNA templates with 1.85 MBq α-32P dCTP (Perkin Elmer, Seer Green, UK), random decanucleotides and Klenow polymerase exo- fragment (Thermo Scientific DecaLabel DNA Labelling Kit, Waltham, MA). The following primers were used to generate the PCR products:
Probes were purified on NICK columns (GE healthcare, Sale, UK) and a specific activity of 2x10^6 cpm was added to the membranes in hybridisation solution and incubated over night at 42° C. Up to 3 probes were used together (two tRNAs and 5S rRNA). The membranes were rinsed three times with 2x SSPE at room temperature and once with 2X SSPE/2%SDS for 15 minutes at 65°. Bound probe was quantified by phosphorimaging.

Connor T.M., Hoer S., Mallett A., Gale D.P., Gomez-Duran A., Posse V., Antrobus R., Moreno P., Sciacovelli M., Frezza C., Duff J., Sheerin N.S., Sayer J.A., Ashcroft M., Wiesener M.S., Hudson G., Gustafsson C.M., Chinnery P.F, & Maxwell P.H. (2017). Mutations in mitochondrial DNA causing tubulointerstitial kidney disease. PLoS Genetics, 13(3), e1006620.

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Northern Blot Analysis of Viral RNA

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For Northern blot analyses, 3 µg of total RNA was separated on formaldehyde-1.5% agarose gel and blotted to Amersham Hybond-NX membrane (GE Healthcare, Chicago, IL, USA), by the capillary method using 20× SSC (3 M NaCl and 0.3 M Na-citrate; pH 7.0). Hybridization was carried out at 65 °C in Church buffer (0.5 M Sodium Phosphate buffer, pH 7.2 containing 1% BSA, 1 mM EDTA, 7% SDS) overnight with the appropriate radioactively labelled probe, washed for 5 min in 2× SSC, 0.1% SDS and for 15 min in 0.5× SSC, 0.1% SDS at the temperature of the hybridization and exposed to an X-ray film. Virus-specific, P32-labelled DNA probes were prepared by using the DecaLabel DNA Labelling Kit (Thermo Fischer Scientific). As a template for Northern blot, probe PCR-amplified and purified product of the cloned region of CVA was used.

Baráth D., Jaksa-Czotter N., Molnár J., Varga T., Balássy J., Szabó L.K., Kirilla Z., Tusnády G.E., Preininger É, & Várallyay É. (2018). Small RNA NGS Revealed the Presence of Cherry Virus A and Little Cherry Virus 1 on Apricots in Hungary. Viruses, 10(6), 318.

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Southern Blot Analysis of Genomic DNA

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The genomic DNAs were digested with restriction endonucleases (5 U µg^–1 DNA, 6 h), fractionated by agarose gel electrophoresis and transferred to Hybond XL membranes (GE Healthcare, Marlborough, MD, USA) using alkaline capillary transfer. The probe was an insert of the CANR4.4 clone (GenBank MK069592) amplified with the universal vector Sp6 and T7 primers. Approximately 100 ng of purified PCR product was radioactively labelled with ³²P-dCTP according to the manufacturer’s protocol (DecaLabel DNA Labelling Kit, Thermo Fisher, Waltham, MA, USA). Southern blot hybridization was performed in 0.25 m sodium phosphate buffer (pH 7.0) supplemented with 7 % (w/v) sodium dodecyl sulphate (SDS) at 65 °C. The membranes were washed with 2× SSC (10× SSC = 1.5 m NaCl, 0.15 m sodium citrate, pH 7.0), 0.1 % SDS (twice for 5 min) and then with 0.2× SSC and 0.1 % SDS (twice for 15 min at 65 °C). The membranes were exposed to a storage phosphor screen, scanned (Typhoon FLA7400; GE Healthcare), and the signal was quantified using Image Quant (GE Healthcare).

Lunerová J., Herklotz V., Laudien M., Vozárová R., Groth M., Kovařík A, & Ritz C.M. (2020). Asymmetrical canina meiosis is accompanied by the expansion of a pericentromeric satellite in non-recombining univalent chromosomes in the genus Rosa. Annals of Botany, 125(7), 1025-1038.

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Telomere Length Measurement by Southern Blot

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Telomere length analysis was performed as described in (34 ) by Southern hybridization of terminal restriction fragments (TRF) produced by digestion with Tru1I restriction endonuclease (Thermo Scientific). A membrane was hybridized overnight at 55°C with telomere probe (Supplementary File S1C). Telomere probe was synthesized by non-template PCR according to (35 (link)) and radioactively labelled with [³²P-dATP] using DecaLabel DNA labelling kit (Thermo Scientific). Telomere signals were visualized using a PhosphoImager FLA-7000 (GE Healthcare). Telomere lengths were calculated by WALTER toolset v2.0 (36 (link)) using default setup with background correction.

Fajkus P., Kilar A., Nelson A.D., Holá M., Peška V., Goffová I., Fojtová M., Zachová D., Fulnečková J, & Fajkus J. (2021). Evolution of plant telomerase RNAs: farther to the past, deeper to the roots. Nucleic Acids Research, 49(13), 7680-7694.

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Northern Blot Analysis of Viral RNA

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For Northern blot analyses 4–5 μg of total RNA was separated on formaldehyde-1.2% agarose gel and blotted to Amersham Hybond-NX membrane (GE Healthcare, UK), by capillary method using 20xSSC. Hybridization was carried out at 65°C in Church buffer (0.5 M Phosphate buffer, pH 7.2 containing 1% BSA, 1 mM EDTA, 7% SDS) overnight with the appropriate radioactively labeled probe, washed for 5 min in 2 × SSC, 0.1% SDS and for 15 min in 0.5 × SSC, 0.1% SDS at the temperature of the hybridization and exposed to an X-ray film.
Virus-specific, P³²-labeled, DNA probes were prepared by using the DecaLabel DNA Labelling Kit (Thermo Fischer Scientific, USA). As a template we used the PCR-amplified and purified product of cloned region of viral genome. The virus piece cloned was a 1663-bp part amplified with GPGV5557F and GPGV7220R for GPGV, a 1324-bp product amplified by RBDV_RNA1F_4082 and RBDV_RNA1R_5406 for RBDV RNA1, and a 927-bp product amplified by GSVsatF72 GSVsatR999 for GSV.

Czotter N., Molnar J., Szabó E., Demian E., Kontra L., Baksa I., Szittya G., Kocsis L., Deak T., Bisztray G., Tusnady G.E., Burgyan J, & Varallyay E. (2018). NGS of Virus-Derived Small RNAs as a Diagnostic Method Used to Determine Viromes of Hungarian Vineyards. Frontiers in Microbiology, 9, 122.

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Northern Blot Analysis of p14 Expression

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To detect p14 6 μg of same RNA as was used to detect miR168 was separated on formaldehyde agarose gels, blotted onto Hybond NX membrane (GE Healthcare) with capillary transfer, UV crosslinked and subjected to hybridization with radiolabeled PCR product of p14 at 65ºC as described previously (39 (link)). Probe was labelled using DecaLabel DNA labelling Kit of Thermo Fisher Scientific according to the manufacturer's instructions. Image was created with phosphorimager screen (Amersham).

Dalmadi Á., Miloro F., Bálint J., Várallyay É, & Havelda Z. (2021). Controlled RISC loading efficiency of miR168 defined by miRNA duplex structure adjusts ARGONAUTE1 homeostasis. Nucleic Acids Research, 49(22), 12912-12928.

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Northern Blot Analysis of Viral RNAs

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For Northern blot analyses, 3 µg of total RNA were separated on a denaturing-formaldehyde 1.5% agarose gel and blotted to Amersham Hybond-NX membrane (GE Healthcare, Chicago, IL, USA) by the capillary method using 20× SSC (3 M NaCl and 0.3 M Na-citrate; pH 7.0). Hybridization was carried out at 65 °C in Church buffer (0.5 M Sodium Phosphate buffer, pH 7.2 containing 1% BSA, 1 mM EDTA, 7% SDS) overnight with the appropriate radioactive probe, washed for 5 min in 2× SSC, 0.1% SDS and for 15 min in 0.5× SSC, 0.1% SDS at hybridization temperature and exposed to an X-ray film. Virus-specific, P³²-labelled DNA probes were prepared using the DecaLabel DNA Labelling Kit (Thermo Fischer Scientific). PCR-amplified and purified products of the cloned regions from all three RNAs were used as a template. Two membranes were prepared and hybridized with RNA2 and RNA3-specific probes. After exposure, the membrane hybridized with the RNA2-specific probe was stripped and rehybridized with the RNA1-specific probe.

Elbeaino T., Kontra L., Demian E., Jaksa-Czotter N., Slimen A.B., Fabian R., Lazar J., Tamisier L., Digiaro M., Massart S, & Varallyay E. (2020). Complete Sequence, Genome Organization and Molecular Detection of Grapevine Line Pattern Virus, a New Putative Anulavirus Infecting Grapevine. Viruses, 12(6), 602.

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Southern Blot Analysis of Cry2AX1 Transgene

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Ten micrograms of genomic DNA samples from four selected T3 progenies (27-13-10, 27-13-16, 27-25-13 and 27-25-24) were digested with EcoRI or NcoI enzymes, which have single recognition site within the T-DNA and fractionated in 0.8 % agarose gel. Gel was denatured, neutralised and the digested genomic DNA was transferred onto Hybond-N+ nylon membrane (Sigma-Aldrich, St. Louis, USA). The transferred DNA was cross-linked at 1200 µJ min-1 for 1 min by using UV crosslinker. Amplicons of internal region of cry2AX1 fragment (800 bp) were generated by PCR and used as probe. The probe DNA was labelled with radioactive α32P dCTP by random priming using Decalabel DNA labelling kit (Thermo scientific Inc. Waltham). Hybridization was carried out for 16 hr at 60° C. After hybridization, the blot was washed for 15 min with 3X SSC + 0.1 % SDS and 2X SSC + 0.1 % SDS. Another washing with 0.5X SSC + 0.1% SDS was given for 10 min. All washings were carried out at 60° C. The blot was washed after hybridization to remove excess radioactive material and the membrane was exposed to X-ray film.

Jadhav M.S., Rathnasamy S.A., Balakrishnan N., Sudhakar D., & Udayasuriyan V. (2020). Study of Expression of Indigenous Bt cry2AX1 Gene in T3 Progeny of Cotton and its Efficacy Against Helicoverpa armigera (Hubner). Brazilian Archives of Biology and Technology, 63.

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Telomere Length Analysis in Plants

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TRF analysis was performed as described in Mozgová et al. (2010) . Plant DNA was extracted from 5-w-old leaves in accordance with Dellaporta et al. (1983) and 400 ng of gDNA was used for the analysis. DNA was digested with 20 U of MseI (NEB) at 37°C overnight, DNA fragments were separated on the 0.8% agarose gel and alkali-blotted onto a Hybond XL membrane (GE Healthcare Life Sciences, Waukesha, WI, USA). Telomere fragments were detected by a radioactively labeled telomeric DNA probe, prepared by nontemplate PCR according to Ijdo et al. (1991) and labelled with [α-32 P] ATP using random decamers (Decalabel DNA labelling kit, Thermo Fisher Scientific). Hybridization was performed overnight at 65°C. Telomeric signals were visualized using a FLA7000 imager (Fujifilm) and quantified as described in Zachová et al. (2013) . Quantification was carried out as follows: the medians of TRF lengths were calculated as Σ(ODi x Li)/Σ(ODi), in which ODi is the signal intensity above the background within intervals i, and Li is the molecular weight (kb) at the midpoint of interval I. In the charts, the bottom and top of the box represent the first and third quartile, respectively, separated by the median, and s.d. intervals above and under the box are the maximum and minimum of the telomere lengths, respectively.

Eekhout T., Dvořáčková M., Pedroza-García J.A., Dadejová M.N., Kalhorzadeh P., Daele H.V.d., Vercauteren I., Fajkus J., & Veylder L.D. (2021). Loss of FAS1 function reveals rescue of an aberrant intra-S-phase checkpoint by the G2/M checkpoint regulator SOG1^1[OPEN].

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