INTRODUCTION OF GENES USING PARTICLE BOMBARDMENT

11. INTRODUCTION OF GENES USING PARTICLE BOMBARDMENT

The initial steps in this experiment involved coating the gold particles with the DNA plasmid. 5 mg of gold beads were weighed into a centrifuge tube and 400 µl of 100% ethanol added. The solution was subjected to an ultrasonic disruptor 10 X per second and flashed at 10000 rpm. The supernatant was sucked off, and dried for 5 minutes under a clean bench. 4 µl of NaOAC and 40 µl of DNA (0.5 µg/µl TE) were added and the solution shaken vigorously. 100 µl ethanol was added and kept at –80 degrees C for 15 minutes. The solution was flashed at 10000 rpm at 4 degrees C and the sup was sucked off. 500 µl of ethanol (1 mgAu/100 EtOH) was added and the mixture subjected to ultrasonic disruptor 3 X per second, using a vortex, and gold beads solution added (10 µl X two times per plate). It was later dried up and bombardment done using a titanium coin.

The titanium coin was washed twice with double distilled water, then washed twice with 70% ethanol, and washed a third time with 100 % ethanol, and the gold particles solution applied on the coin and allowed to dry ready for bombardment.