1.
Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), vortex thoroughly (for about 1 min). If you use Eppendorf caps, make sure to hold them closed tightly while vortexing. If you do not meet the the Nimblegen requirements in terms of absorbtion values, you can repeat this step to increase purity.
2.
Spin for 5 min at 13,000 rpm at RT to separate the phases. Carefully remove the upper aqueous phase and transfer it to fresh 1.5 ml microcentrifuge tube.
3.
Add approximately 6 µg of nuclease-free Glycogen (0.5µl = 10µg of the Invitrogen solution) together with a half volume of 7.5M NH
4
OAc, and 2.5 volumes of ice-cold
95% ethanol. Vortex the mixture thoroughly and immediately centrifuge for 20min at
13,000 rpm and RT.
4.
Remove the supernatant carefully and discard. Overlay the pellet with 0.5ml of ice cold 70% ethanol. Centrifuge 10 min at 13,000 rpm and remove and discard the supernatant. You should see a pellet at this stage.
5.
Dry the cDNA at RT for 10 min to evaporate residual alcohol and dissolve the pellet in 14µl water (take 1 µl for nanodrop and 1 µl to agarose gel). Make sure you meet the Nimblegen requirements (4 µg of cDNA at at least 250 ng/µl, A260/280>1.7,
A260/230>1.5, mean size > 400bp).
6.
If the quantity of cDNA is not sufficient, repeat steps V and VI, resuspend the cDNA pellet in the 12 µl remaining from the first cDNA synthesis (so as not to increase the volume) and add 2µl of H2O to bring the volume up to 14 µl for the gel/Nanodrop sampling.