The precipitation is undertaken to concentrate the cleaned dscDNA ready for efficient in vitro transcription

2. Precipitate each cleaned ds-cDNA sample, with the following reagents:

2.5X volumes 100% cold ethanol 375 |L

0.5X volume of 7.5 M ammonium acetate 75 |L

5 mg/mL glycogen 4 |L

3. Mix by tapping tubes or vortex briefly.

4. Centrifuge immediately at RT and maximum speed for 20 min.

5. A pellet of precipitated ds-cDNA will have formed at the bottom of the tubes. Keeping samples on ice, carefully remove the supernatant.

6. Wash the ds-cDNA pellet from salts by adding 500 |L of cold 80% ethanol and then centrifuge at maximum speed for 5 min. Remove the supernatant (see Note 33).

7. Repeat cold 80% ethanol wash of step 6 once.

8. Carefully pipet away the ethanol, ensuring the ds-cDNA pellet is left intact. Briefly spin the tubes to pool the residual ethanol and use a pipette to remove.

9. Air dry the ds-cDNA pellets. This will take between 5 to 10 min (see Note 34).

10. Add 12 mL of DEPC H2O to each pellet, resuspending by gentle pipetting.

11. Either proceed to the next stage, in vitro transcription (Subheading 3.6.), or freeze the cleaned, ds-cDNA by placing tubes on dry ice and then storing in a -80°C freezer (see Notes 35 and 36).

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