John M. S. Bartlett.pdf - Bio-Nica.info

44 Pearson
7. Add 60 µL of chloroform/isoamyl alcohol (241) and vortex vigorously.
8. Place on ice for 15 min.
9. Microfuge at 12,000g for 5 min and transfer the upper phase to new microfuge tube.
10. Add 200 µL of ice-cold isopropanol mix and store at –20°C for 30 min.
11. Microfuge at 12,000g for 15 min and discard supernatant.
12. Resuspend pellet in 200 µL of extraction buffer.
13. Repeat steps 3 through 9.
14. Wash pellet with 400 µL of cold 70% ethanol.
15. Microfuge at 12,000g for 5 min and discard supernatant.
16. Carefully remove last traces of ethanol from tube (folded sterile swab or kimwipe works
well).
17. Resuspend in 100 µL of distilled water and incubate at 50°C for 15 min to dissolve RNA
(see Note 4).
4. Notes
1. Blood stored at room temperature or 4°C should be mixed thoroughly prior to aliquots
being removed.
2. Frozen blood samples should be allowed to thaw completely and mixed thoroughly before
aliquots being removed. Although freezing lyses red blood cells, the red cell lysis step
should still be performed to efficiently remove hemoglobin from the sample. Repeated
freeze/thaw cycles should be avoided.
3. Buffy coat contains two to four times the amount of white blood cells per volume compared
to fresh blood. Therefore, it is advisable to use only 50 µL of buffy coat diluted with 50 µL
of phosphate-buffered saline as starting material for this protocol.
4. Repeat pipetting through a narrow gauge tip can help this process.
References
1. Chomczynski, P. and Sacchi. N. (1987) Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.