Nd-to-end PCR for detecting whether all plastomes are in transformed state. The process has been employed previously for investigating that all plastid genomes are in transformed state and no wild-type plastid genome is left [90,91]. For this goal, a pair of primers was used which gave good benefits for both wild-type and transgenic lines with different amplicon sizes. Sense primer, oli253, was located inside plastome outside trnN and anti-sense primer oli252 was situated within the trnR. The sequences of those primers had been: oli253 (five -GATCCGAGCCATAGAATTTC-3) and oli252 (5 -AGACAGCGACGGGTT CTCTG-3). The typical PCR reaction situations have been used and Tm from the primers was 52 C. The positions of these primers and anticipated fragment sizes are shown in (Figure 1). four.6. Western Blot Immunoblot analysis was carried out following the procedure with minor modifications as described [87]. Protein was extracted in the leaves of transplastomic and wild-type plants. To extract the total soluble protein from transplastomic and wild-type plants, approximately 100 mg of leaves have been ground thoroughly in liquid nitrogen and then homogenized within a protein extraction buffer containing: 0.5 M sorbitol, ten mM ethylene glycol tetra acetic acid (EGTA), ten mM sodium orthovanadate (Na3 VO4), ten mM sodium JTP-117968 In Vitro fluoride (NaF), five (v/v) polyvinylpyrrolidone, 25 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid-BTP (HEPES-BTP) (pH 7.6) and five further components [0.5 (w/v) bovine serum albumin (protease totally free, A-3294 obtained from Sigma, St. Louis, MO, USA), 1 mM dithiothreitol, 0.five mM phenylmethyl sulfonyl fluoride, five /mL leupeptin, and 0.five /mL pepstatin A] had been added just before use. The homogenized samples were centrifuged at 14,000 g for 10 min at four C as well as the supernatants had been collected as soluble fraction. Soluble