NH4Ac and EtOH precipitation

NH4Ac and EtOH precipitation of DNA

Add NH4Ac (10M stock or solid) to the sample for a final concentration of 2.5M, mix (spin at 4℃, 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 10min) 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(0.5ml) MFT(1.5ml) Corex  ORT 250ml

DNA 105μl 210ul  315μl  2.1ml  9ml  60.0ml

NH4Ac  35μl  70μl  105μl  0.7ml  3ml  12.6g

EtOH 350μl 700μl 10μl  7.0ml 30ml 165.0ml

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