Dig high prime kit

Manufactured by Roche

Sourced in Germany, Switzerland

The DIG-High Prime kit is a laboratory product designed for the labeling of nucleic acids with digoxigenin (DIG). The kit provides reagents and protocols for the non-radioactive detection of DNA or RNA targets in various applications such as Southern, Northern, in situ hybridization, and dot-blot analysis.

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18 protocols using dig high prime kit

Extraction and Analysis of Fungal and Plant DNA

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Total DNAs from fungi were extracted following the method of Raeder and Broda (Raeder and Broda, 1985 (link)), using mycelium collected from a PDB culture incubated at 28°C and 200 rpm for 2 days. DNA isolation from tomato roots was performed with the cetyltrimethylammonium bromide (CTAB) extraction method (Dellaporta et al., 1983 (link)). Total RNA was extracted using TRIZOL® reagent (Invitrogen Life Technologies, Carlsbad), following the instructions of the manufacturer.
For Southern analyses, 10 μg of genomic DNA was SacI-digested, electrophoresed on a 0.7% agarose gel and transferred to a Hybond-N⁺ membrane (Amersham Biosciences AB, Uppsala, Sweden). The phleomycin gene was labeled using the DIG High Prime kit (Roche, Penzberg, Germany), following the manufacturer's instructions, and used as probe. Hybridization, washes and detection were carried out as previously described (Tijerino et al., 2011 (link)).

Pérez E., Rubio M.B., Cardoza R.E., Gutiérrez S., Bettiol W., Monte E, & Hermosa R. (2015). The importance of chorismate mutase in the biocontrol potential of Trichoderma parareesei. Frontiers in Microbiology, 6, 1181.

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Labeling and Hybridization of DNA Probes

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DNA probes were labelled with digoxigenin using the DIG-High Prime kit (Roche, Basel, Switzerland). Positively charged nylon membranes (Roche, Basel, Switzerland) were pre-hybridized with 0.5 mg/mL sonicated and denatured salmon sperm DNA (Roche, Basel, Switzerland) in a 20 mL prehybridization solution (2× SSPE, 0.5% Blotto, 1% SDS, 10% dextran sulphate) at 65 °C for 4–6 h. Labeled DNA was added, and hybridization continued for another 12–16 h. The hybridized membranes were sequentially washed with 2× SSC (0.3 M NaCl in 30 mM Sodium Citrate) and 0.1% SDS at room temperature for 5 min twice and with 0.1× SSC and 0.1% SDS at 68 °C for 15 min twice as well. Detection was performed with an Antidigoxigenin-Alkaline Phosphatase conjugate antibody (Roche, Basel, Switzerland) and CDP-Star (Perkin Elmer, Inc., Waltham, MA, USA) according to the instructions provided by the manufacturer. The membranes were then exposed for different times to X-ray Agfa films (Agfa HealthCare, Mortsel, Belgium).

Cebrián J., Martínez V., Hernández P., Krimer D.B., Fernández-Nestosa M.J, & Schvartzman J.B. (2021). Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates. Biology, 10(11), 1195.

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Maize Genomic DNA Isolation and McGISH

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The total genomic DNA of Z. mays (inbred line Mo17) and Z. perennis (CIMMYT accession No. 9475) were isolated from young leaves using a modified 2× CTAB method (Fu et al. 2015 ), and then labeled with probes with a DIG-High Prime Kit and a Biotin Nick Translation Kit (Roche, Basel, Switzerland), respectively, according to the manufacturer’s instructions. The McGISH technique was performed according to the methods of Iqbal et al. (2019) (link). The McGISH images were visualized via a phase video microscope (Olympus BX61, Tokyo, Japan) equipped with a charge-coupled-device camera (700 mm CCD) and via Image Pro Plus 6.0 (Media Cybernetics, Silver Spring, USA).

Yan X., Li Y., Wu Z., Li Y., Wen X., Li X., He R., Yang C., Zhao Y., Cheng M., Zhang P., Sam E.K., Rong T., He J, & Tang Q. (2020). Analysis of the genitor origin of an intergeneric hybrid clone between Zea and Tripsacum for forage production by McGISH. Breeding Science, 70(2), 241-245.

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Plant DNA Extraction and Blot Analysis

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Total genomic DNA (~ 10 μg) extracted from plant tissue was resolved on 1.5% agarose gels, transferred overnight by capillary action onto Hybond N⁺ membrane (Amersham) and UV cross-linked. Probes were produced by PCR using a DIG-high prime kit (Roche Applied Science) and primers βC1F (5′-AGACCCGGGATGACGATCAGATATAATAACA-3′)/ βC1R (ACGTCGACTCACACACACACTTTCGTACA-3′) for betasatellite and (qPCRChV F (5′-AGTTCATATAGGGAAGGTTATGTGTA-3′) / qPCRChV R (5′- GGACAAGGAAGAACATCACACTATTC-3′) for virus. Membranes were hybridized and washed as described previously [22 (link), 23 (link)] and hybridization was detected by colorimetric detection with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, toluidine-salt (Roche Applied Science).

Shahid M.S., Shafiq M., Raza A., Al-Sadi A.M, & Briddon R.W. (2019). Molecular and biological characterization of Chilli leaf curl virus and associated Tomato leaf curl betasatellite infecting tobacco in Oman. Virology Journal, 16, 131.

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Labeling DNA Probes for Membrane Hybridization

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DNA probes were labeled with digoxigenin using the DIG-High Prime kit (Roche Applied Science). Membranes (Zeta-Probe GT membranes, Bio-Rad) were pre-hybridized in a 20 ml pre-hybridization solution (2× SSPE, 0.5% Blotto, 1% SDS, 10% dextran sulfate, and 0.5 mg/ml sonicated and denatured salmon sperm DNA) at 65°C for 4–6 h. Labeled DNA was added, and the hybridization lasted for 12–16 h. Hybridized membranes were sequentially washed with 2× SSC and 0.1% SDS at room temperature for 5 min twice and with 0.1× SSC and 0.1% SDS at 68°C for 15 min twice as well. Detection was performed with an antidigoxigenin-alkaline phosphatase-conjugated antibody (Roche Applied Science) and CDP-Star (PerkinElmer Life Sciences) according to the instructions provided by the manufacturers as described before [29 (link)]. All experiments were performed twice to confirm the results obtained were reproducible.

Castán A., Fernández-Calleja V., Hernández P., Krimer D.B., Schvartzman J.B, & Fernández-Nestosa M.J. (2017). Analysis of DNA topology of EBV minichromosomes in HEK 293 cells. PLoS ONE, 12(11), e0188172.

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DNA Probe Labeling and Hybridization

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DNA probes were labelled with digoxigenin using the DIG-High Prime kit (Roche). Membranes were pre-hybridized in a 20 ml pre-hybridization solution (2x SSPE, 0.5% Blotto, 1% SDS, 10% dextran sulphate and 0.5 mg/ml⁻¹ sonicated and denatured salmon sperm DNA) at 65°C for 4–6 h. Labeled DNA was added and hybridization lasted for 12–16 h. Hybridized membranes were sequentially washed with 2x SSC and 0.1% SDS at room temperature for 5 min twice and with 0.1x SSC and 0.1% SDS at 68°C for 15 min twice as well. Detection was performed with an antidigoxigenin-AP conjugate antibody (Roche) and CDP-Star (Perkin Elmer) according to the instructions provided by the manufacturer. Quantification of autoradiograms was performed using a ImageJ64 software.

Cebrián J., Monturus E., Martínez-Robles M.L., Hernández P., Krimer D.B, & Schvartzman J.B. (2014). Topoisomerase 2 Is Dispensable for the Replication and Segregation of Small Yeast Artificial Chromosomes (YACs). PLoS ONE, 9(8), e104995.

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GISH Technique for Chromosomal Analysis

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For genomic in situ hybridization (GISH), chromosomal preparations were
made from D9(E4) and D9(E6)C2B3 by conventional methods [22 (link)]. Total genomic DNA for use as probes was extracted
from gerbil splenocytes and mouse myeloma cells with a DNeasy Blood & Tissue Kit
(Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. The probes were labeled
with biotin-16-dUTP by using a Biotin-High Prime Kit (Roche, Tokyo, Japan; for mouse DNA)
and with DIG-11-dUTP by using a Dig-High Prime kit (Roche; for gerbil DNA) according to
the manufacturer’s protocol. Signals were detected with Alexa Fluor 488-streptavidin
(Invitrogen, Tokyo, Japan; green fluorescence for mouse DNA) and
anti-digoxigenin-rhodamine (Roche; red fluorescence for gerbil DNA). GISH was conducted as
reported previously [23 (link)].

UKAJI T., HASHIMOTO M, & KAI O. (2015). Conditioned medium from gerbil—mouse T cell heterohybridomas improved antibody secretion. Experimental Animals, 64(2), 199-205.

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Digoxigenin-labeled DNA Probe Generation

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To generate the digoxigenin (DIG)-labeled probes, DNA fragments corresponding to parts of the gerE and gin element adjacent of the junction sites were amplified from the B. cereus chromosomal DNA with the primer sets PA246/PA306 and PA309/PA310, respectively. PCR products were gel-purified and labeled by the incorporation of DIG-11-dUDP using the DIG High Prime kit (Roche, Mannheim, Germany). Five micrograms of chromosomal DNA from B. cereus was digested by HindIII overnight, separated by 1.2% agarose gel electrophoresis, and transferred onto a Hybond-N⁺ membrane (GE Healthcare, NJ, USA) by the capillary method using 10 × SSC. Hybridization and detection were performed using the DIG-labeled probes, anti-DIG antibody conjugated to alkaline phosphatase (Roche), and a nitro-blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate solution (NBT/BCIP; Roche). Blot images were cropped and converted to gray scale, using Paintgraphic2 (SOURCENEXT).

Abe K., Shimizu S.Y., Tsuda S, & Sato T. (2017). A novel non prophage(-like) gene-intervening element within gerE that is reconstituted during sporulation in Bacillus cereus ATCC10987. Scientific Reports, 7, 11426.

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Labeling DNA Probes with Digoxigenin

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DNA probes were labeled with digoxigenin using the DIG-High Prime kit (Roche). Membranes (Zeta-Probe GT membranes, Bio-Rad) were pre-hybridized in a 20 ml pre-hybridization solution (2× SSPE, 0.5% Blotto, 1% sodium dodecyl sulphate (SDS), 10% dextran sulphate and 0.5 mg/ml⁻¹ sonicated and denatured salmon sperm DNA) at 65°C for 4–6 h. Labeled DNA was added and hybridization lasted for 12–16 h. Hybridized membranes were sequentially washed with 2× Saline sodium citrate (SSC) and 0.1% SDS at room temperature for 5 min twice and with 0.1× SSC and 0.1% SDS at 68°C for 15 min twice as well. Detection was performed with an antidigoxigenin-AP conjugate antibody (Roche) and CDP-Star (Perkin Elmer) according to the instructions provided by the manufacturer.

Castán A., Hernández P., Krimer D.B, & Schvartzman J.B. (2017). The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks. Nucleic Acids Research, 45(17), 10089-10102.

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Papaya Viral DNA Detection via Southern Blot

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Papaya DNA was extracted with the E.Z.N.A. High Performance Plant DNA Maxi Kit (Omega) and used for Southern blotting. CP-gene probes were prepared by amplification with specific primers CP1 and CP2 (Table 1) and labeled with Digoxigenin-11-dUTP using DIG-High-Prime Kit (Roche). About 60 μg of genomic DNA was digested with Hind III and BamH I, separately. Electrophoresis of the genomic DNA was performed in 1.0% agarose gel. The DNA was transferred to nylon membranes (GE Healthcare) using the capillary siphon blot method²⁷, and then fixed by UV-crosslinking (Scientz Biotechnology, LTD) at 243 nm for 45 min. Hybridization was performed overnight at 42 °C in a hybridization incubator (Model 2000 Robbins Scientific). Immunological detection was made with anti-Digoxigenin-AP following the product manual, and recorded with an EC3 imaging system (UVP, LLC).

Jia R., Zhao H., Huang J., Kong H., Zhang Y., Guo J., Huang Q., Guo Y., Wei Q., Zuo J., Zhu Y.J., Peng M, & Guo A. (2017). Use of RNAi technology to develop a PRSV-resistant transgenic papaya. Scientific Reports, 7, 12636.

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