RNA Isolation Using GeneElute Total Mammalian RNA miniprep Kit

Requirements:

As per Kit Specifications

Preparation of Reagents:

1. Lysis Solution/2-ME Mixture: Add 10µl of 2-mercaptoethanol for 1ml of Lysis solution (Add 2-ME to a volume of Lysis solution sufficient for that day’s use only)

2. Wash Solution2: Dilute the concentrate with 10ml (10 prep kit) of absolute ethanol

Procedure:

1. Use rapidly growing cells for total RNA isolation (cell confluency 80-90%)

2. Trypsinize and pellet down the cells

3. Wash the cells with PBS

4. Vortex pellet to loosen cells

5. Add 250µl of Lysis solution/2-ME mixture for up to 5X10⁶ cells or 500µl of Lysis solution/2-ME mixture for up to 1X10⁷ cells

6. Vortex or pipette thoroughly until all clumps disappear

7. Pipette the lysed cells into a GenElute Filtration Column (blue insert with a 2ml receiving tube)

8. Centrifuge at 13,000rpm for 2min

9. Discard the filtration column

10. Add equal amount of 70% ethanol solution to filtered lysate

11. Vortex or pipette thoroughly to mix

12. Pipette up 700µl of the lysate/ethanol mixture into a GenElute Binding Column (colorless insert with a red o-ring seated in a 2ml receiving tube)

13. Centrifuge at 13,000rpm for 15sec

14. Discard the flow-through liquid, but retain the collection tube

15. Repeat step 12-14 if lysate/ethanol mixture volume is more than 700µl

16. Pipette 500µl of wash solution-1 into the column and centrifuge at 13,000rpm for 15sec

17. Transfer binding column into a fresh collection tube

18. Discard the flow-through and original collection tube

19. Pipette 500µl of wash solution-2 into the column and centrifuge at 13,000rpm for 15sec

20. Discard the flow through

21. Pipette 500µl of wash solution-2 into the column and centrifuge at 13,000rpm for 2min

22. Centrifuge the column for an additional 2 min to dry the column

23. Transfer binding column into a fresh 2ml collection tube

24. Pipette 50µl of elution solution into binding column and centrifuge at 13,000rpm for 1min

25. Repeat elution with a second 50µl elution solution

26. Discard binding column and check RNA concentration and purity by spectrophometric analysis