Ammonium acetate precipitation of DNA - Help is neede for ammonium acetate precipitation of DNA (Jul/06/2007 )
I am in need of the exact protocol for the ammonium acetate precipitation of DNA. As I need DNA for transfection of cells. I tried to do it but all was in vein.
Sambrook says 10M ammonium acetate stock and final conc should be 2.0-2.5M.
Can anybody tell me is it made in ddH2O or ethanol? How much to add to my DNA? I mean in terms of micro liter and Molar conc.
Thanks in advance.
Prepare it in H2O, add 1/5 of the total volume or 1:4 stock solution to DNA.
agree with genehunter-1.
that means if my DNA sup is 100 ul then i add 25ul Ammonium acetate from 10 M stock.
but if you want to use the DNA for ligation purpose its better to use Na-acetate,
at least 3 EtOH-70 washes are needed to remove decently the ammonium acetate present in the pellet.
Prepare as above, when precipitate the DNA is better to use 2-2.5 volume of cold ethanol absolute or isopropanol (I keep mine at -20C), add the salt (ammonium acetate, sodium acetate or lithium chloride) and incubate in cold. (better if left at -20C O/N), but if you are in hurry can left at -80C 1hour. Then wash 2-3X with etOH 70%. The must important thing is to dry very well the pellet, etoh inhibits pcr reactions. Then resuspend pellet in 0.5X TE (to much edta inhibits some reactions, so keep as low as possible).
I found adding salt to DNA first, then EtOH or isopropanol works better.
Thanks a lot to genehunter-1, T. reesei, fred_33, merlav and genehunter-1.
So, now the points i got from your replies are:
- Use Na acetate ppt for ligation
- Atlease 3 70% ethanol washes
- Dry pellet fully.