Five micrograms of total genomic DNA were fractionated on a 1% (w/v) agarose gel at 34 V overnight (ca. 12 hrs). The DNA was transferred onto a positively charged nylon membrane (GE Healthcare, Piscataway, NJ, USA) using 20× SSC for at least 8 hrs and UV crosslinked (Green and Sambrook, 2012 ). Probes for virus detection were PCR amplified using the primers listed in Supplementary Table 1, which targeted the replicase (AC1) and movement protein (BC1) genes of each virus species. Probes were labelled with use of a digoxigenin (DIG) DNA labelling kit (Roche). The subsequent steps were performed as described by Beyene et al. (2015) .
Dig dna labelling kit
Manufactured by Roche
Sourced in Switzerland, Germany
The DIG DNA Labeling Kit is a tool used to label DNA molecules with a digoxigenin (DIG) probe. The kit provides reagents and protocols for the direct labeling of DNA through incorporation of DIG-labeled nucleotides. This allows for the subsequent detection of the labeled DNA using anti-DIG antibodies.
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Lab products found in correlation
Positively charged nylon membrane, by GE Healthcare (1 mentions)
Revertra ace qpcr rt master mix, by Toyobo (1 mentions)
Rlm rt pcr kit, by Roche (1 mentions)
Thunderbird sybr qpcr mix without rox, by Toyobo (1 mentions)
Dig luminescent detection kit, by Roche (1 mentions)
Positively charged nylon membrane, by Merck Group (1 mentions)
Alexa fluor 546 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 633 conjugated goat anti guinea pig igg, by Thermo Fisher Scientific (1 mentions)
Anti dig fitc, by Roche (1 mentions)
6 protocols using dig dna labelling kit
1
Genomic DNA Fractionation and Viral Probing
2
Quantitative RT-PCR and Northern Blot Analysis
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RLM RT-PCR kit was used to clone intact mRNA with a 7-methyl guanosine cap structure (Roche).
For quantitative RT-PCR, complementary DNAs (cDNAs) were synthesized with total RNAs (800 ng per reaction) with ReverTra AceqPCR RT Master Mix (Toyobo, Japan). Each cDNA was diluted 20-fold, and used as a template for qRT-PCR analysis using Thunderbird SYBR qPCR Mix without ROX (Toyobo). The genes act and tef were used as internal standards50 (link). The relative expression level of each gene was determined using the 2−ΔΔCt method51 (link).
Northern blot analyses were conducted using standard methods52 . The ORF of the Mr-OPY2 was used as the probe, which was labelled with DIG-DNA Labelling Kit (Roche). Northern blot and qRT-PCR analyses were repeated three times.
For quantitative RT-PCR, complementary DNAs (cDNAs) were synthesized with total RNAs (800 ng per reaction) with ReverTra AceqPCR RT Master Mix (Toyobo, Japan). Each cDNA was diluted 20-fold, and used as a template for qRT-PCR analysis using Thunderbird SYBR qPCR Mix without ROX (Toyobo). The genes act and tef were used as internal standards50 (link). The relative expression level of each gene was determined using the 2−ΔΔCt method51 (link).
Northern blot analyses were conducted using standard methods52 . The ORF of the Mr-OPY2 was used as the probe, which was labelled with DIG-DNA Labelling Kit (Roche). Northern blot and qRT-PCR analyses were repeated three times.
3
Non-radioactive Northern Hybridization for CircRNA
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Non-radioactive northern hybridization was performed with the purified PCR fragment (>200 nt) as the probe, which spanned the corresponding circRNA junction site. Probe preparation was followed with the DIG DNA labelling kit (Roche, Basel, Switzerland) according to manufacturer's instructions.
4
Southern Blot Analysis of At-NPR1 Gene
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20 μg of genomic DNA was digested with EcoRI. Following electrophoresis on 1.0% agarose gel, DNA was stained with ethidium bromide for 30 s and examined under UV light. The digested DNA fragments were transferred to positively charged nylon membranes (Pall Gelman, USA) by alkaline transfer method as described by Sambrook [81] . 500 bp PCR product of At-NPR1 gene was used to prepare probe for Southern analysis. The probe was labelled with DIG (dig-oxigenin) using a DIG-DNA labelling kit (Roche, Germany) following manufacturer’s protocol. Southern blots containing the genomic DNA were hybridized to the DIG-labelled At-NPR1 probe at 60 °C for 24 h. The hybridized probe was immuno-detected using anti-DIG–AP conjugate in the presence of the chromogenic substrate NBT/BCIP (nitro-blue tetrazolium chloride/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt), following the manufacturer’s protocol.
5
Northern Blot Analysis of lincRNA-p21
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LincRNA-p21 probe was labelled with digoxigenin (DIG) using a DIG DNA Labelling Kit (Roche). Total RNA extracted from AML12 cells and run on a 1% denatured agarose gel, transferred to positively charged nylon membranes (Millipore) followed by cross-linking through UV irradiation. The membrane was then hybridized with (DIG)-labelled probe overnight. The detection was performed using a DIG luminescent detection kit (Roche) according to the manufacturer’s instructions.
6
FISH Probe Generation and Hybridization
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~20-kb FISH probes were generated using a long-range PCR kit (Encyclo Plus PCR (Evrogen)) by PCR-amplification of 4 tiling genome fragments covering either the region 2 L:16964000–16982000 or 2 L:17310000–17328000, with the use of primer pairs provided in the Supplementary Table 1 . 1 µg of template DNA for hybridization was labelled by random primed synthesis with the DIG DNA labelling kit (Roche) or by ChromaTide Alexa Fluor 546–14-dUTP (Life Technologies). Probes were further combined and hybridized with S2 cells as described previously23 (link). For NL or FISH probe detection, as the primary antibodies we used guinea pig polyclonal anti-LBR26 (link) (1:1000), or rabbit polyclonal anti-lamin Dm051 (link) (1:500) and sheep polyclonal anti-DIG-FITC (1:500, Roche). As the secondary antibodies we used Alexa Fluor 633-conjugated goat anti-guinea pig IgG (Invitrogen), or Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen) and Alexa Fluor 488-conjugated goat anti-FITC IgG (Invitrogen).
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