Southern blot analysis was carried using the DIG DNA Labeling and Detection Kit (Roche Applied Sciences, Mannheim, Germany) according to the manufacturer's instructions. Genomic DNA was isolated from randomly selected PCR-positive in vitro grown plants using cetyltrimethylammonium bromide (CTAB) method (Soni and Murray, 1994 (link)). About 20 μg of genomic DNA was digested overnight at 37°C with HindIII (New England Biolabs, USA) for which there is a single recognition site in the T-DNA of pCAMBIA2301, separated by electrophoresis on 0.8% (w/v) agarose gel at 40 V for 6 h, and transferred to Hybond-N+ nylon membrane (Roche) and fixed by cross-linking in a STRATA-LINK™ UV cross-linker. The blots were hybridized with digoxigenin (DIG)-labeled gusA-specific probe generated using a PCR DIG Probe Synthesis Kit (Roche Applied Sciences, Mannheim, Germany). Hybridization and detection were carried out using a DIG Luminescent Detection Kit for Nucleic Acids (Roche Applied Sciences, Mannheim, Germany), according to the manufacturer's instructions.
Dig luminescent detection kit for nucleic acids
Manufactured by Roche
Sourced in Germany, United Kingdom
The DIG Luminescent Detection Kit for Nucleic Acids is a laboratory product designed for the detection of nucleic acids. It utilizes a digoxigenin (DIG) labeling system and a luminescent detection method. The kit provides the necessary reagents and protocols for the sensitive and specific detection of labeled nucleic acid targets.
Automatically generated - may contain errors
Lab products found in correlation
Pcr dig probe synthesis kit, by Roche (3 mentions)
Dig dna labeling and detection kit, by Roche (2 mentions)
Hybond n membrane, by Cytiva (2 mentions)
Hybond, by Cytiva (2 mentions)
Positively charged nylon membrane, by Roche (2 mentions)
Hybond n nylon membrane, by Roche (1 mentions)
Hindiii, by New England Biolabs (1 mentions)
Nylon membrane, by GE Healthcare (1 mentions)
Dig dna labeling mix, by Roche (1 mentions)
Positively charged membrane, by Roche (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Superscript first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Ultra hyb oligo hybridisation buffer, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Lightcycler 480 instrument, by Roche (1 mentions)
Dig application manual for filter hybridization, by Roche (1 mentions)
11 protocols using dig luminescent detection kit for nucleic acids
1
Southern Blot Analysis of Transgenic Plants
2
Detecting HBV DNA Replication Intermediates
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The isolation and determination of HBV DNA replication intermediates were performed as previously described. These HBV DNA replication intermediates (HBV DNA RI) were resuspended in 30 μL of tris‐ethylene diamine tetraacetic acid buffer. The samples were separated by 1% agarose and transferred to Hybond‐N+ membrane (Amersham Biosciences, Bucks, UK). The membrane were probed with digoxigenin‐labeled full‐length HBV DNA sequence and then analyzed using the DIG Luminescent Detection Kit for Nucleic Acids (Roche, Germany).
3
HBV DNA Replication Intermediate Analysis
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HBV DNA replication intermediates were extracted and determined according to the protocol described in previous literatures13 (link). Samples were resuspended in 30 μL of tris-ethylene diamine tetraacetic acid (TDTA) buffer and separated with 1% agarose gel. HBV replication intermediates samples were transferred to Hybond-N+ membrane (Amersham Biosciences, UK) and detected using digoxigenin-labeled full-length HBV DNA sequence as probe following further detection with the DIG Luminescent Detection Kit for Nucleic Acids (Roche, Germany).
4
Xa21 Gene Integration in Banana
5
Detecting Transgene Copy Numbers by Southern Blot
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Transgene copy numbers were detected by Southern blot hybridization analysis. Approximately 12 μg of total DNA was digested and blotted onto nylon membranes (GE Healthcare, UK). A 477-bp LRP gene fragment was amplified using 5′-ATGGGTGTTTTCACATATG-3′ and 5′-TCAATTGTATTCAGGATGG-3′ to generate the hybridization probe by DIG DNA Labeling Mix (Roche, Switzerland). Detection was performed using a DIG Luminescent Detection Kit for Nucleic Acids (Roche, Switzerland). The detailed procedure was according to the manual.
6
Southern Blot Analysis of AtEFR Transgenic Plants
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Southern blot analysis was performed as per the method described by Tripathi et al. [23 (link)]. Briefly, 10 μg of genomic DNA from each sample was restricted with BamH1 for 12 h. The DNA samples, including plasmid pBIN19g-35S::AtEFR and genomic DNA sample from a control non-transgenic plant, were run for a 0.8% agarose gel at 50 V. The gel stained with GelRed® was viewed under ultraviolet light to confirm the digestion. The restricted DNA was denatured, then blotted onto a positively charged membrane (Roche Diagnostics, West Sussex, UK) and fixed using ultraviolet cross-linking. The blots were then hybridized with a digoxigenin (DIG) PCR-labeled 635-bp AtEFR-specific probe. Hybridization and probe detection was performed using a DIG Luminescent Detection Kit for Nucleic Acids (Roche Diagnostics, UK) as per the manufacturer’s protocol.
7
Transgene Integration and Copy Number Analysis in Cassava
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Southern blot analysis was done to confirm integration of transgenes into the cassava genome and to determine copy number of transgene according to DIG DNA Labeling and Detection Kit (Roche Applied Sciences, Mannheim, Germany) following manufacturer’s instructions. Genomic DNA was extracted from leaves of in vitro grown plants using cetyltrimethylammonium bromide (CTAB) method as described by Soni and Murray [19] (link). A total of 20 μg genomic DNA was digested overnight at 37 °C with PmlI (New England Biolabs, USA) for which there is a single recognition site in the T-DNA of pCam:Ostlp, separated by electrophoresis on 1.0% (w/v) agarose gel followed by depurination, denaturation and neutralization steps and transferred to a positively charged nylon membrane (Roche Applied Sciences, Mannheim, Germany) by capillary transfer. After cross-linking, the blots were hybridized with DIG-labeled nptII-specific probe generated using a PCR DIG Probe Synthesis Kit (Roche Applied Sciences, Mannheim, Germany). Hybridization and detection were performed using a DIG Luminescent Detection Kit for Nucleic Acids (Roche Applied Sciences, Mannheim, Germany), following the instructions of the manufacturer.
8
Southern Blot Validation of Clta Integration
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The correct integration was validated using Southern Blot. The Sothern Blot probe was synthesized according to manufacturer’s protocol using the PCR DIG probe synthesis kit (Roche) using the following primers: Clta-NdeI-for 5’-GTA TCA GCC AAG CCA TTG GT-3‘ and Clta-NdeI-rev: 5‘-TGG GCA TGG AAA AAG AAG TC-3‘ . DNA from animal tissue was isolated, digested with ScaI at 37°C overnight, following the recommendations of the manufacturer, separated by agarose gel electrophoresis, transferred to a positively charged nylon membrane, and finally detected using the Dig luminescent Detection Kit for Nucleic Acids (Roche) and an x-ray film developer machine.
9
Comprehensive Transcriptome Analysis Protocol
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Total RNA was extracted from fresh tissues and cultured cells using Trizol (15596–026, Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s protocol. Superscript First-Strand cDNA Synthesis Kit (18080–051, Invitrogen, Carlsbad, CA) was used to reverse transcribe 500 ng total RNA into cDNAs. Quantitative RT-PCR was performed using SYBR Premix Ex Taq II kit (DRR081A, TAKARA,Otsu, Shiga, Japan) on LightCycler 480 System (Roche, Basel, Switzerland). Northern blot assays were performed using DIG Luminescent Detection Kit for Nucleic Acids (11363514910, Roche, Basel, Switzerland) in the light of the manufacturer’s instructions. The primer sequences were listed in Supplementary Table 1 .
10
Northern Blot Analysis of Small RNAs
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Small RNA fraction (size ≤ 200 nts) was separated from total RNA samples, isolated from respective transgenic and vector control leaves according to Lu et al. [47 (link)]. Equal amount of small RNA (nearly 36 μg) was separated on 15% polyacrylamide-urea gel and electroblotted onto the positively charged nylon membrane (N+, Bright Star-Plus, Ambion) by following the protocol of Rio [48 (link)]. Transferred small RNA molecules were immobilised on the membrane by EDC cross-linking as described by Paul and Hamilton [49 (link)]. Synthesized oligonucleotide (21 nt), complementary to HaAce1-amiR1, was labelled with DIG-dUTP using 2nd generation oligonucleotide 3'- end labelling kit (Roche). Hybridization was carried out in ULTRAhyb-oligo hybridisation buffer (Ambion) containing 0.3 nanomoles of DIG end-labelled probe at 37°C for 16 hours. Membrane was subsequently washed with 2X SSC + 0.1% SDS at 28–32°C. Further steps were carried out according to the instructions provided by DIG Luminescent Detection Kit for nucleic acids (Roche, version 07).
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