mRNA in situ hybridization using digoxigenin-labelled probes was performed to determine the spatial expression patterns of BxML1 in B. xylophilus. A 501-bp gene was used to generate/synthesize probes. Antisense and sense DIG-labelled probes were prepared separately by unidirectional PCR with these templates. In situ hybridization was performed as described previously [58 (link)] using a DIG High Prime RNA Labeling and Detection Starter Kit I (Roche Diagnostics). Finally, the samples were observed under an Axio Image M2 microscope (Zeiss).
Dig high prime rna labeling and detection starter kit 1
Manufactured by Roche
The DIG High Prime RNA Labeling and Detection Starter Kit is a laboratory equipment product designed for the labeling and detection of RNA. It provides the necessary components for the non-radioactive labeling of RNA using digoxigenin (DIG) and the subsequent detection of the labeled RNA.
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3 protocols using dig high prime rna labeling and detection starter kit 1
1
Spatial Expression of BxML1 in B. xylophilus
2
In situ Hybridization of B. xylophilus
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A 179 bp PCR product was amplified from B. xylophilus (AMA3) cDNA using forward and reverse primers. These amplicons were used as the template in a unidirectional PCR to produce sense and antisense DIG‐labelled probes. In situ hybridization was performed as described previously (de Boer et al., 1998 (link)) using a DIG High Prime RNA Labeling and Detection Starter Kit I (Roche Diagnostics). Finally, the samples were observed under an Axio Image M2 microscope (Zeiss).
3
In situ Hybridization of B. xylophilus
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An 810‐bp PCR product was amplified from B. xylophilus AMA3 cDNA. Antisense and sense DIG‐labelled probes were prepared separately by unidirectional PCR with these templates. The primers we used are listed in Table S1 . In situ hybridization was performed as described previously (de Boer et al., 1998 (link)) using a DIG High Prime RNA Labeling and Detection Starter Kit I (Roche). Finally, the samples were observed under an Axio Image M2 microscope.
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