Reagents:

• ddH₂O

• Phenol, water-saturated and buffered (pH 7.5)

• Chloroform

• G25 column

• Linear acrylamide

• 3M Sodium Acetate pH 7.5 and pH 5.2

• Ethanol

• T7 polymerase with 5x reaction buffer and DTT

• Nucleosides

• RQ1 RNase-free DNase

Protocol:

A. Prepare template

1. Adjust volume of template PCR or linearized plasmid to 200 µl with ddH₂O

2. Phenol/Chloroform extract 3x with 100 µl P/C pH7.5

3. Pass through G25 column

4. Add:

   1. 1 µl linear acrylamide

   2. 30 µl Sodium Acetate pH 7.5

   3. 600 µl 100% Ethanol

5. Mix, precipitate at -80°C for 15 minutes.

6. Spin 12 minutes at 14,000 rpm

7. Wash pellet with 70% Ethanol

8. Wash pellet with 100% Ethanol

9. Dry pellet for 10 minutes

10. Dissolve pellet in 11 µl ddH₂O

11. Use 1 µl template to determine concentration.

12. Use 1.5 µg template in 10 µl ddH₂O for transcription reaction.

B. In vitro transcription reaction

1. Make reaction mix:

   1. 38 µl ddH₂O

   2. 20 µl 5x Buffer

   3. 10 µl 100 mM DTT

   4. 5 µl 10 mM rGTP

   5. 5 µl 10 mM rATP

   6. 5 µl 10 mM rUTP

   7. 5 µl 10 mM rCTP

   8. 1 µl RNase Inhibitor

   9. 1 µl T7 polymerase

2. Vortex, spin briefly

3. Add 10 µl template DNA, mix, spin briefly

4. Incubate for 1 hour at 40°C

C. Destroy DNA, purify RNA

1. Add 1 µl RQ1 RNase-free DNase

2. Incubate for 15 minutes at 40°C

3. Add 106 µl ddH₂O

4. Sequentially add the following:

   1. 200 µl Phenol (water-saturated)

   2. 40 µl Chloroform

5. Vortex, spin for 5 minutes at 14,000 rpm

6. Pass the upper aqueous phase through G25 column

7. Precipitate with:

   1. 1 µl linear acrylamide

   2. 30 µl 3M Sodium Acetate pH5.2

   3. 600 µl 100% Ethanol

8. Mix, precipitate at -80°C for 15 minutes.

9. Spin 15 minutes at 14,000 rpm

10. Wash pellet with 70% Ethanol

11. Wash pellet with 100% Ethanol

12. Dry pellet for 10 minutes

13. Dissolve pellet in 11 µl ddH₂O

14. Use 1 µl to determine RNA concentration

Svetlana Mikheeva, 13 August 1999