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Preparation of poly(A)+ RNA

Purification of poly(A)+ RNA from total RNA

0. (Optional)
Before using Oligotex-dT30, I recommend to wash Oligotex-dT30 to remove fine particle of latex. To wash the latex, transfer appropriate amount of Oligotex-dT30 (300 ul of the suspension of Oligotex per 1mg of total RNA) into a microfuge tube. Spin for 3 min at 12,000 rpm. Discard the supernatant. Gently suspend the latex in the same volume of 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% SDS. Spin again. Gently suspend the latex in the same buffer.

1. Add 1 mg of total RNA dissolved in RNase-free water to 300 ul of the Oligotex-dT30 suspension.

2. Incubate for 3 minutes at 65 C. Chill on ice.

3. Add 0.2 volume of 5M NaCl. Incubate for 10 minutes at 37 C.

4. Centrifuge for 3 minutes at 15,000 rpm. Discard the supernatant.

5. Suspend the pellet in 1 ml of washing buffer (10mM Tris-HCl pH7.5, 1mM EDTA, 0.5M NaCl, 0.1% SDS).

6. Centrifuge for 3 minutes at 15,000 rpm. Discard the supernatant.

7. Suspend the pellet in 300 ul of RNase-free water containing 0.1% SDS.

8. Incuabte for 5 minutes at 65 C. Chill on ice.

9. Centrifuge for 3 minutes at 15,000 rpm. Transfer the supernatant into new microfuge tube.

10. Carry out phenol-extranction, chloroform-extraction and ethanol precipitation by standard procedure. Rinse the pellet with 75% ethanol. Dissolve the poly(A)+ RNA in 10 ul of RNase-free water.



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