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- Add one volume of phenol:chloroform:isoamyl alcohol (25:24:1) to your sample, and vortex or shakeby hand thoroughly for approximately 20 seconds.
- Centrifuge at room temperature for 5 minutes at 16,000 × g. Carefully remove the upper aqueous phase, and transfer the layer to a fresh tube. Be sure not to carry over any phenol during pipetting.
- Proceed to Ethanol Precipitation, below.
|Glycogen (20 μg/μL)||1 μL|
|7.5 M NH4OAc||0.5 × volume of sample|
|100% ethanol||2.5 × volume of sample +NH4OAc|
- Add the following reagents to the aqueous phase, in the listed order in above table
- Place the tube at –20°C overnight to precipitate the DNA from the sample. Note: If you wish to continue with the protocol, place the tube in dry ice or at –80°C for at least 1 hour.
- Centrifuge the sample at 4°C for 30 minutes at 16,000 × g to pellet the cDNA.
- Carefully remove the supernatant without disturbing the cDNA pellet.
- Add 150 μL of 70% ethanol. Centrifuge the sample at 4°C for 2 minutes at 16,000 × g. Carefully remove the supernatant.
- Repeat Step 3 once. Remove as much of the remaining ethanol as possible.
- Dry the cDNA pellet in a SpeedVac® for 2 minutes or at room temperature for 5–10 minutes.
- Resuspend the cDNA pellet in 300 μL of TEN buffer by pipetting up and down 30–40 times.
- Centrifuge briefly to collect the sample, and place the tube on ice.