Amersham hybond n nylon membrane

The full-length McAGO4-A, McAGO4-Like and McDRM2 cDNA PCR products were subcloned into the pMAL-C2X expression vector [71 (link)]. Bacterial growth and protein induction were performed as described by the manufacturer (Novagen). Following induction of protein expression in the cells with 0.3 mM/100 mL isopropyl-β-D-thiogalactoside (IPTG), and washing with and suspending in PBS solution, 5 mL bacterial aliquots (12 mg/mL) were stored at −80 °C.
Oligonucleotides were synthesized and biotin-labeled at the 3′ end (Sangon Biotech; www.sangon.bioon.com.cn, accessed on 26 July 2021). Standard reaction mixtures [72 (link)] were used for EMSA: 2 μL 10 × binding buffer, 1 μL 50% glycerol, 1 μL 100 mM MgCl₂, 1 μL 1% NP-40, 2 μg protein, 2 μL biotin-labeled oligonucleotides (0.1μM). The reaction mixtures were incubated at room temperature (25 °C) for 20 min and then electrophoretically separated on 12% native polyacrylamide gels and transferred to an Amersham Hybond^TM N⁺ nylon membrane (GE Healthcare) in TBE buffer [72 (link)] at 380 mA at 4 °C for 40 min. After UV cross-linking, biotin-labeled DNA was detected using a LightShift Chemiluminescent EMSA kit (Pierce, Shanghai, China). The primer and probe sequences are listed in Supplementary Table S1.

Genomic DNA (20 μg) was digested with SpeI and AgeI for 24 h at 37 °C. The digested samples were subjected to agarose gel electrophoresis in 0.8% agarose gel (15 cm × 15 cm) at 30 V for 18 h, followed by 50 V for 3 h. A digoxigenin (DIG)-labeled DNA marker (DNA molecular weight marker III, Roche, Germany) was run together for the size analysis. The DNA fragments in the agarose gel were transferred onto a nylon membrane (Amersham hybond^TM-N⁺ nylon membrane, GE Healthcare, Chicago, IL, USA) using standard blotting methods. After the transfer, the DNA fragments on the membrane were cross-linked using CL-1000 Ultraviolet Crosslinker (Spectrum chemical, New Brunswick, NJ, USA). The nylon membrane was subjected to hybridization with a 10 ng/mL DIG-labeled probe specific to the hapX gene. The DIG-labeled hapX probe was generated by PCR using hapX-probe-fwd and hapX-probe-rev primers (Supplementary Table S1) followed by DIG labeling using a labeling kit (Random Primed DNA Labeling Kit, Roche, Rotkreuz, Switzerland). The DIG-labeled DNA was visualized by the NBT/BCIP reaction after binding with 150 mU/mL of Anti-DIG-AP conjugate (Roche, Rotkreuz, Switzerland).

Northern analysis was performed with M. tuberculosis H37Rv total RNA as per the standard protocol43 . Briefly, 5 to 8 μg of total RNA isolated from log phase cultures was resolved on 1.2% or 1.5% agarose gel containing 6% formaldehyde. RNA was transferred to Amersham Hybond-N + nylon membrane (GE Healthcare) overnight using upward capillary transfer. It was then cross-linked to membrane by exposure to UV and incubated with denatured radiolabeled probe after pre-hybridization. [α-P³²] dCTP-labeled DNA probes were generated near the 5′ and 3′ ends of the operon. Hybridization was done overnight at 42 °C, followed by several membrane washes. Finally, the blot was exposed to phosphorimager screen which was then scanned using phosphorimager ImageQuant to visualize the hybridization signals.

For Southern blotting analysis, 40 μg rubber tree genome DNA was digested with EcoRI, PstI, XbaI and HindIII respectively. After resolved on 0.8% agarose gel, the blot was transferred onto Amersham Hybond-N⁺ nylon membrane (GE Healthcare, USA) and cross-linked under 0.12 J/cm² UV irradiation. Then the blot was hybridized at 42°C with Digoxigenin labeled probe for 12h. After 2 stringent washes at 68°C, the signal was detected using DIG Nucleic Acid Detection Kit (Roche, USA).

For Northern hybridization analysis, dsRNAs were separated on a 0.7% agarose gel and run in 0.5x TBE buffer for 16 h at 50 V. The gel was then denatured in 0.1 M NaOH for 30 min and neutralized in 0.1 M Tris-HCl (pH 8.0) for 30 min. The RNAs were transferred to an Amersham Hybond-N+nylon membrane (GE Healthcare) by capillary action. Digoxigenin (Dig)-labeled DNA preparation, prehybridization, and hybridization were performed as the user manuual (GE Healthcare). The probe-RNA hybrids were detected using a CDP-Star kit (GE Healthcare). Probe 1 was amplified using the specific primers BRV1S1-F (5′-CCAGGGCAGTTGCTACAGTCAT-3′) and BRV1S1-R (5′-TTGCCGCAGAGGGGAATC-3′), probe 2 was amplified using the specific primers BRV1S2-F (5′-TCCCCGAGCAACTCAAAAACC-3′), and BRV1S2-R (3′-GCACGATTTAGTGCTGCGGTTT-3′), and probe 3 was amplified using the primers BRV1-p1-F (5′-GCAATAAAAAGCACAGCCGG-3′) and BRV1-p1-R (5′-TGTTGTGTTATTTGGTATGTTGATCG-3′).