Dig high prime dna labeling and detection starter kit 2

Genomic DNA was extracted from tail of the transgenic/control mouse by using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China). PCR assay was performed by using the primers shown in Table 1. The 1226 bp PCR product contains partial K14 promoter and partial ovine β-catenin DNA sequence. The PCR procedure: 5 min at 95°C, 35 cycles of 30 s for 95°C, 30 s for 58°C and 72°C for 1 min, 72°C for 10 min and hold at 4°C forever. The further southern blot assay, digestion: the BstEⅡ was selected for digested the DNA (60°C, 20 h), which is obtained from mouse tail. The probe (739 bp) amplified by primer (F6 and R6 in Table 1) across promoter and β-catenin sequence, was labeled with the PCR digoxigenin probe synthesis kit (Roche). The DIG-High Prime DNA Labeling and Detection Starter KitⅡ was used for washing and hybridization (Roche, Basel, Switzerland).

To determine the location of optrA, S1 nuclease-pulsed field electrophoresis (S1-PFGE) and Southern blot were performed following the methods described previously (Barton et al., 1995 (link); Rosvoll et al., 2010 (link)). Briefly, bacterial cells harvested from fresh culture were embedded in agarose gels and then digested by S1 nuclease (TaKaRa, Dalian, China). Salmonella Braenderup H9812 chromosomal DNA was digested with XbaI (TaKaRa, Dalian, China) and used as the DNA size marker. CHEF electrophoresis was performed using the same conditions described elsewhere (Rosvoll et al., 2010 (link)). Following transfer to Hybond N + membranes (Amersham Biosciences, United States) by capillary blotting for more than 20 h, blots were hybridized with digoxigenin (DIG)-labeled optrA-specific probe using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Sciences, Germany) following the manufacturer’s instructions.

Total RNAs were extracted from shrimp gills by using the mirVanaP^TMP miRNA isolation kit (Ambion, USA) according to the manufacturer’s protocol. After separation on a denaturing 15% polyacrylamide gel containing 8 M urea, the small RNAs were transferred to a Hybond-N+ membrane (Amersham Biosciences, Buckinghamshire, United Kingdom). Subsequently, the RNAs were cross-linked under UV light (Ultra-Violet Products Ltd., USA). The membrane was prehybridized in DIG Easy Hyb Granules buffer (Roche, Basel, Switzerland) for 30 min and then hybridized with digoxigenin (DIG)-labeled probes completely complementary to WSSV-miR-22 (5′-UUUCCUUACGAAUGAAAAGUAA-3′) at 42 °C overnight. The DIG-labeled U6 probe (5′-GGGCCATGCTAATCTTCTCTGTATCGTT-3′) was used as a control. Immunological detection was performed using the DIG High Prime DNA labeling and detection starter kit II (Roche, Basel, Switzerland).

Genomic DNA isolation and Southern blotting were performed as previously described (Ding & Butler, 2007). Briefly, 5 ml of cell culture (YPD medium or YPGal medium) was washed twice with water and then resuspended in lysis buffer (100 mM Tris‐HCl pH 8.0, 50 mmol/L EDTA, 1% SDS, 100 mM NaCl, 2% Triton X‐100). Cells were disrupted using a bead beater (Biospec). The supernatant was mixed with 7 mol/L ammonia acetate (pH 7.0). Genomic DNA was extracted using chloroform and precipitated with isopropanol. DNA pellets were then washed, dried, and resuspended in water. Next, 1 μg of DNA was used for PCR. Genomic DNA (20 μg) was treated with RNase A and digested with StuI and SacII overnight for Southern hybridization analysis. Southern blotting was performed using a DIG High Prime DNA Labeling and Detection Starter Kit II (Roche), and the experimental procedure was carried out according to the manufacturer's manual. The DIG‐labeled DNA probe was synthesized by PCR using primers CDp1045 and CDp612 (Table S1).

The SmsHSP24.1 coding region (NdeI + NotI) was first cloned into Gateway compatible entry vector (pL12R34-Ap) under constitutive cauliflower mosaic virus promoter (CaMV 35S) (KpnI + NdeI) and nopaline synthase terminator (Nos) (NotI + SacI) and then transferred into the plant transformation vector pMDC100 (Invitrogen, United States) containing nptII (kanamycin-resistant) gene as a plant selection marker (Supplementary Figures 3D-G). Agrobacterium strain, EHA105 was used for plant transformation, and 21-days-old cotyledonary leaves were used as starting material. Kanamycin (100 mg/l) was used at a 10-day interval to select transformed lines. The putative transgenic lines were screened by PCR using nptII and SmsHSP24.1 gene-specific primers. For Southern blot analysis of putative transgenic lines, approximately 20 μg genomic DNA was digested with the restriction enzyme, NdeI. DNA fragments were separated on a 0.8% agarose gel and blotted onto the Hybond^TM N⁺ nylon membrane (GE Healthcare Limited, United Kingdom). Subsequently transferred N⁺ nylon membrane was hybridized with a 500 bp of SmsHSP24.1 gene-specific DIG-labeled probe (PCR DIG Probe Synthesis Kit, Roche, Germany). The blot was then washed and detected according to the instructions of the manufacturer (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche, Germany).