<p>Bio443</p><p>Expt 2: Procaryotic and Eucaryotic RNA Isolation </p><p>RNA Isolation 1. Obtain 2 cell pellets (1 procaryotic and 1 eucaryotic). 2. Add 0.5 ml of Trizol reagent and resuspend the pellet. 3. Transfer to a microfuge tube and incubate at room temperature for 5 minutes 4. Add 0.1 ml of chloroform and shake the tubes by hand for 15 seconds 5. Incubate at room temperature for 10 minutes 6. Centrifuge the samples at 12,000 g for 10 minutes 7. Transfer the colorless aqueous phase to a new tube and precipitate the RNA with 0.25 ml of isopropanol. 8. Incubate the samples at room temperature for 10 minutes. 9. Centrifuge the samples at 12,000 g for 10 minutes. 10. Remove the supernatant carefully by pouring it off. 11. Wash the pellet with 1 ml of cold 75% ethanol. 12. Mix the sample gently and centrifuge at 12,000 g for 7 minutes. 13. Remove the supernatant carefully by pouring it off. 14. Air dry the pellet for 10 minutes. 15. Resuspend the pellet in 40 ul of DEPC water. 16. Heating the RNA for 5-10 minutes at 55C may be necessary to completely resuspend the pellet. 17. Put the RNA on ice. RNA Sample Preparation 1. Determine the concentration of your RNA samples on the Nano-drop. </p><p>2. Determine the volume of your samples needed for 2 ug of RNA</p><p>3. Set up the denaturation reaction for your samples according to the table below.</p><p>Eukaryotic (ul)</p><p>RNA ______</p><p>Volume Formaldehyde Loading Dye 3.0</p><p>DEPC water ______</p><p>Total 10.0</p><p>4. Heat the samples at 70C for 10 minutes. 5. Following denaturation, place the samples immediately on ice.</p><p>Electrophoresis of Samples 1. Rinse the electrophoresis unit out with DEPC water 2. Open and place the precast gel in the electrophoresis unit. 3. Fill the buffer chambers with 1X MOPS Running buffer. 4. Load your samples (2 per lab) plus the markers 4. Run the gel at about 80V until the bromophenol blue dye has travelled about 3 cm. 5. Wearing gloves, remove the gel from the unit.</p>