NH4Ac and EtOH precipitation of DNA
Add NH4Ac (10 M stock or solid) to the sample for a final concentration
of 2.5 M, mix (spin at 4 C, transfer the supernatant to a new
tube; optional spin for extra purification of the DNA), add 2.5
volumes of EtOH, mix, and spin this time keeping the pellet (watch
the liquid as pouring it down the drain to make sure that pellet
does not come loose). Turn the tube upside down on a paper towel
to allow the EtOH to drain well. Give the pellet a 70 % EtOH
rinse (carefully and a little slowly add the EtOH by a DPTP: if
the pellet comes loose, spin it again), drain the tube again,
and dry the pellet briefly (under the vacuum for 5 to 10 min)
before resuspending the DNA in 10:2 TE (almost always TE except
doing a digest or ligation in which case the DNA is resuspended
in H2O:low salt in the resuspension buffer is important). Often
multiple NH4Ac/EtOH precipitation are desired. If the DNA is
particularly important or if it is at a very low concentration
i.e. <0.5 µg/ml, it is best to include a waiting period
before each spin: at least one hour (overnight is better) and
at either room temperature or 4 C. EtOH and salt reduce the solubility
of DNA, consequently, it is best to drain the EtOH well, wiping
the inside of the tube with a Chimwipe (but, do not touch the
pellet)
Sample Volume of Table
MFT(0.5 ml) MFT(1.5 ml) Corex ORT 250ml
DNA 105 ul 210 ul 315 ul 2.1 ml 9 ml 60.0 ml
NH4Ac 35 ul 70 ul 105 ul 0.7 ml 3 ml 12.6 g
EtOH 350 ul 700 ul 1050 ul 7.0 ml 30 ml 165.0 ml
Optional spin check:
Min/rpm 15/14K 20/14K 10/14K 25/9K
Min/rpm 20/14K 20/14K 20/14K 20/14K 20/14K 25/9K