2020/05/27

2020-04-24 TRIzol + RNeasy Plus Mini Kit

For IsoSeq gDNA contamination need to be removed

1. Tissue in 800 ul TRIzol & homogenize

2. Incubate for 5 min
3. Chloroform 160 ul, mix
4. Incubate for 3 min
5. ⟳ 15 min, 12,000 g @4°C
6. Aqueous phase + 450 ul 100% isopropanol
7. Incubate 10 min @4°C
8. ⟳ 10 min, 12,000 g @4°C
9. Pellet dissolved in 150 ul H2O
10. Add RLT 500 ul
11. Apply to gDNA eliminator Spin Column
12. ⟳ 30 sec, 8,000 g @>20°C
13. Save flow-through(FT) + 600 ul 70% EtOH
14. 700 ul samples apply to RNeasy column
15. ⟳ 30 sec, 8,000 g @>20°C
16. Discard FT
17. RW1 700 ul, ⟳ 30 sec, >8,000 g @>20°C, discard FT
18. RPE 500 ul, ⟳ 30 sec, >8,000 g @>20°C, discard FT
19. RPE 500 ul, ⟳ 2 min, >8,000 g @>20°C, discard FT
20. New 2ml collecting tube, ⟳ 60 sec, >8,000g @>20°C, discard tube
21. New 1.5ml tube, add 52 ul RNase-free water, sit for 3 min
22. ⟳ 1 min, >8,000 g to elute