Differential Centrifugation – Isolation of Ricketssia from Red Abalone Post-Esophegus Tissue

Post-esphagus (PE) tissue was isolated from one control abalone (12:6-1; 0.077g PE) and three “hot” abalone (11:8-8, -9, -10; 0.1606g PE, 0.126g PE, 0.1205g PE) by Lisa. Control abalone PE was homogenized in 0.5mL TriReagent and stored @ -80C. The three “hot” abalone PE were individually homogenized in ice cold 5mL of 1x Tris Sucrose Buffer (TSB). pH = 7.4 until the entire tissue was fully homogenized, including the difficult connective tissue.

Samples were transferred to 15mL conical tubes and spun at 250g for 10mins @ 4C. The supe was transferred to a 30% Percoll-TSB gradient. The pellet was placed into 1mL TriReagent, vortexed and stored @ -80C (Hot PE Pellet). 25uL from the pellet and the supe were saved for qPCR analysis.

The gradient was spun at 25000g for 2hrs at 4C in a Sorvall T21 centrifuge in a SL-50T rotor with the “SoftSpin” setting on and the brake turned off.

Below is a link to a slide show of the sample at various stages of preparation, including images of what the gradient looked like after the final spin.