A DIG probe targeting VviTPS10 was obtained through PCR amplification of an 862 bp internal region of the coding sequence followed by DIG labeled as described in the DIG Application Manual for Filter Hybridization (Roche, Germany) and diluted to 8.2 ng/mL in DIG Easy Hyb. The same probe solution was used for both Southern and Northern blotting at the appropriate temperatures described in the DIG Application Manual for Filter Hybridization (Roche, Germany).
For Southern blot analysis genomic DNA was isolated from MA, SB, and SH using the method described by Lovato et al. (2012) (link), followed by single digests of 10 μg gDNA using BamHI, EcoRI and XbaI restriction enzymes (Thermo Fisher Scientific, United States). Southern blotting was performed as described in the DIG Application Manual for Filter Hybridization (Roche, Germany).
Total RNA was isolated from ±100 mg tissue for EL-18 and EL-26 stages from MA, SB, and SH using the method described by Reid et al. (2006) (link). RNA was selectively purified using the RNeasy Mini kit (Qiagen) according to the RNA clean-up protocol described in the product manual. RNA samples were separated on a 1.2% formaldehyde agarose (FA) gel followed by Northern blot analysis according to the DIG Application Manual for Filter Hybridization (Roche, Germany).
For Southern blot analysis genomic DNA was isolated from MA, SB, and SH using the method described by Lovato et al. (2012) (link), followed by single digests of 10 μg gDNA using BamHI, EcoRI and XbaI restriction enzymes (Thermo Fisher Scientific, United States). Southern blotting was performed as described in the DIG Application Manual for Filter Hybridization (Roche, Germany).
Total RNA was isolated from ±100 mg tissue for EL-18 and EL-26 stages from MA, SB, and SH using the method described by Reid et al. (2006) (link). RNA was selectively purified using the RNeasy Mini kit (Qiagen) according to the RNA clean-up protocol described in the product manual. RNA samples were separated on a 1.2% formaldehyde agarose (FA) gel followed by Northern blot analysis according to the DIG Application Manual for Filter Hybridization (Roche, Germany).
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