Difference between revisions of "Extraction of RNA"

Revision as of 15:43, 23 July 2018

Extracting RNA post stimulation and collection

EXAMPLE: Starting w/ four samples (before, 6hr, 12hrs, 24hrs)

1. Use the hood in the hallway, remove any stuff that shouldn’t be in there (ie old chemical bottles)
2. Work VERY sterile, we have RNAses on us
3. Clean hood with ethanol, replace bench paper (new bench paper is kept on top of fridge in hallway)
4. 1.7 ml tubes for extracted RNA
5. Chloroform [toxic], isopropyl (in Ju Chen’s cabinet- key is kept in his bay)
6. Clean gloves with ethanol (do this every time before you touch something), pre-clean tube rack, clean pipettes
7. Vortex samples (5 seconds) if frozen thaw first
8. Add 200 ul of chloroform
9. Don’t touch chloroform to trizol in tube, pipette from a distance. Trash tip in waste bottle *label the waste bottle when finished*
10. Vortex for 15 seconds each tube
11. Set on bench for 2-3 minutes (set a timer)
12. Centrifuge in the cold room (max speed for 15 minutes) This seperates RNA from everything else in the cell. Don’t want to contaminate RNA with cell particles.
13. Label the new 1.7 ml tubes *make sure both sets of tubes stay in order the entire time*
14. Keep only the upper colorless layer (RNA is here, about 400 ul) and pipette into new corresponding tube. Careful to not disturb the interface between the two layers. Use a 1000 ul and 200 ul tip to take up the 400 ul (maybe 450 ul)
15. Change tips between each tube
16. If you think you touched the interphase take away the tip and re-centrifuge the tube
17. Once the upper layer is in the new tube, add 500ul of isopropyl alcohol to each tube
18. Use a 25ml pipette to aliquot some in a tube (instead of the whole bottle)
19. Invert tubes three times then set at room temperature for 10 minutes
20. Centrifuge the tubes for 15 minutes at max speed (in the cold room) a pellet will form that’s the RNA we will remove from the supernatant
21. Remove all the supernatant from the tube after centrifuge (450 ul)
22. Do NOT disturb the tiny pellet. Tip the tube 45 degrees and use your glove to see the pellet, keep your eye on the pellet while you draw up liquid,
23. Clean RNA with 70% ethanol (in 50 ml tube on bench)
24. If you run out create more from the 100% bottle found under the sink (35 ml of 100% ethanol and rest is milli Q water
25. Vortex each to clean (few seconds)
26. Centrifuge in cold room at max speed for 10 minutes
27. After centrifuging remove the ethanol one tube at a time
28. Use the bigger pipette to get majority of the liquid and the smaller pipette to get the small amount of liquid remaining
29. Close tube after removing liquid so sample doesn’t dry
30. After all the tubes have the ethanol removed, leave the tubes open for no more than 5 minutes
31. Add 30 ul of milli Q water (on bench in 50 ml tube DEPC) slowly push the water everywhere in the bottom of the tube, different than flushing, cycle the water a few times. Close tube.
32. If there is leftover ethanol still in the tube, dry it before adding water
33. Re-dissolve pellet on heat block in the Ju Chen bench area
34. Set heat block for 60 degrees celsius
35. Remove the timer on the setting (should read “--:--”)
36. Remove “mix” and wait for it to heat up
37. Place on heat block for 10 minutes
38. Place finished tubes in -20 fridge in the lab