you are given 1 gm of fish muscle and 2 ml of bacterial culture. devise a plan to extract mitochondria from muscle and and DNA from bacteria (only flow chart)
Answers
Explanation:
Human/rat/mouse tissue was minced and homogenized in isolation buffer [320 mM sucrose, 5 mM Tris (hydroxymethyl) methylaminoethane sulfonic acid (TES), 1 mM EGTA, pH 7.2]. The homogenate was then centrifuged at 1000 g for 5 min at 4 °C. The supernatant was collected and the pellet was resuspended in isolation buffer followed by second round of homogenization and centrifugation as mentioned earlier. The supernatants from both the steps were then pooled and centrifuged at 8,500 g for 10 min at 4°C. The crude mitochondrial fraction obtained in the pellet was resuspended in minimum volume of isolation buffer (~200 µl), overlaid on 6 % (w/v) ficoll solution, and centrifuged at 75,000 g for 30 min at 4°C, to remove the myelin content. The pellet was then resuspended in ~150 µl reconstitution buffer (250 mM sucrose, 10 mM TES, pH 7.2) and stored at -80 °C.