啤酒酵母(S. cerevisiae)mRNA的提取

Materials2 ml size Sarstedt screw cap tubes

BIO-RAD Disposable Chromatography Column （BIO-RAD cat. #732-6008）

Oligo dT cellulose

RNase-free water

5.0 M NaCl

0.5 M EDTA

1.0 M Tris, pH 7.4

10% SDS

70% EtOH

Before starting protocol

1. Measure out 75 mg of oligo dT cellulose into a 2 ml Sarstedt screw cap tubes.

2. Make fresh buffers:

Note: Amounts are for 2 preps; scale up as necessary.

♦ 2X NETS may need to be heated to go into solution.

♦ 1X ETS should be heated to 65 ℃ for use.

Protocol

1. Resuspend at least 2 mg （up to 4mg） of total RNA in 750 l RNase-free water. In general, inputting more total RNA will yield more polyA RNA. This protocol is most successful with inputs of at least 2 mgs. （Less than 1mg of total RNA input is NOT recommended for this protocol.）

2. Wash the oligo dT cellulose 3X with 750 l 1X NETS. （Spin down gently between washes and aspirate off the wash.）

3. Aspirate off final 1X NETS wash and resuspend the cellulose in 750 l 2X NETS.

4. Incubate the RNA at 65 !C for 10 minutes.

5. Add the RNA to the cellulose and mix on rotator for 1 hour at room temperature.

6. Prepare a BIO-RAD Disposable Chromatography Column （BIO-RAD cat. #732-6008） by washing it out once with 750 l 1X NETS.

7. Gently pour the RNA/cellulose mixture into the column and allow it to settle by gravity. （DO NOT MIX）

8. Gently wash the column 3X with 750 l 1X NETS. （Pipette down the side of the column without disrupting the cellulose.）

9. Elute the mRNA into a fresh Eppendorf tube by pipetting 650 l of 65 !C 1X ETS directly and forcibly into the oligo dT cellulose. Repeat 1X into a second tube.

10. Add 65 l 3M NaAcetate to all samples. Fill the remainder of the tube with room temperature isopropanol （~700 l）. Mix well by inverting several times.