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mRNA Purification from MOLT-4 Cells
Contributor: Cungui Mao
Date: Jul. 9, 1996
1. Grow cells to the density of 1x107 cells per ml
2. Collect cells by centrifugation at 2000rpm(GS3 rotor) speed for 10 min
at 4 oC
3. Suspend cells in PBS(10ml per 440 ml culture)
4. Add 200 ml of RNA STAR-60 to cell suspension
5. Shake it vigorously
6. Set it on at room temperature for 5 min
7. Add 0.2 ml chloroform per ml RNA STAR, shake vigorously for 20 seconds
8. Set it on bench for 3 min
9. Centrifuge it at 9000rpm(GS3 rotor) for 20 min at 10 oC
10. Collect upper layer into the baked corox tubes(glass)
11. Add 0.6 volume of isopropyl alcohol, mix it
12. Centrifuge at 12000rpm (Th rotor) for 20 min at 4oC
13. Aspirate supernatant
14.Wash the pellet with 75% EtOH in DEPC-treated water once
15. Air dry pellet for 5min at room temperature
16. Add DEPC-treated water to dissolve RNA by vortexing
17. Measure the concentration of the total RNA(2.5ug/ul, 1.68 of purity)
18. Purify mRNA by running through the mRNA isolation columns from Stratagen
twice
20. Get 32 ug mRNA from 1mg of total RNA, the purity is 1.95.
21. Concentrate mRNA by precipitation in ethonal at -20oC overnight
22. Check the intergration of mRNA by denatured gel