Northern Blotting Analysis of Jatropha Seeds

For Northern blotting, 15 μg of RNA was isolated from immature jatropha seeds and leaves and fractionated on 1.2% agarose-formaldehyde denaturing gel (Qiagen RNeasy Mini handbook). The RNA was blotted onto Hybond-N⁺ nylon membranes (Amersham Pharmacia) and stained for visualization of the RNA bands [17 (link)]. The KAR cDNA probe generated using RT-PCR was labelled with [32P]-dCTP (GE Healthsciences). Pre-hybridization was for 3 hours and hybridization was for 16 hours at 65°C (Techne, Staffordshire UK). Filters were washed first (20 min) in buffer A (2 × SSC + 0.1% SDS) and then Buffer B (20 min) in (1 × SSC + 0.1% SDS) and lastly (30 min) in buffer C (0.5 × SSC + 0.1% SDS) at 65°C. The bound probe was detected by exposing filters to KODAK Biomax MS Autoradiography Film using exposure cassettes at -80°C.