Size marker

A Qiagen DNeasy Plant Mini Kit was utilized to express genomic DNA from 0.1-0.2 g powdered root tissue. The spectrophotometer Shimadzu UV-mini 1240 was used to assess and calculate the quantity and quality of DNA. A commercial set of 10 random 10-mer primers was obtained (Thermo Scientific). PCR amplifications were carried out in a 25 μL reaction mixture with 10 ng of template DNA, 1X Taq polymerase buffer and 1 U of Taq polymerase, and 2.5 mM MgCl2, 1 μM dNTP, 1 mM primer. Amplifications were done in a TC-3000 Thermal Cycler. The cycle programmed was made up of a preliminary denaturation step at 94°C for 5 min, followed by 40 cycles of 94°C for 1 min, 30°C for 1 min, 72°C for 1 min, and a final elongation at 72°C for 5 min. The PCR products were kept on a 1 % agarose gel with ethidium bromide (0.5 μg/mL) and all digital photographs were taken by the UVP GelDoc-It 310 Imaging System. A 1 kb DNA ladder was used as size marker (Fermentas). 10-mer oligonucleotide primers of 60-70% GC content were benefitted from during the monitoring C. sativus genome for the modification. A negative control with no DNA template was also performed in each PCR amplification to validate the absence of any contamination.