2) Adjust the NaCl concentration to 100 mM and chelate any Mg++ with an equimolar amount of EDTA.
3) Add 1/10 volume of 100 mM spermine (generally, a final concentration of 5 mM is sufficient)
4) Incubate on ice for at least 15 min.
5) Centrifuge for 15 min. at 4oC, transfer supernatant to a new tube and save until you are certain that the DNA precipitated.
6) Dissolve spermine precipitate (DNA precipitate) in 50 ul of 0.6 M NaOAc (high salt is required to dissolve the spermine precipitate.
7) Vortex intermitantly for at least 15 min and maintain the solution at room temperature or 37oC.
8) add 50 ul of water and 250 ul of ethanol, incubate on ice for at least 15 min.
9) Centrifuge for 30 min. in cold, discard supernatant
10) Rinse DNA once with 75% ethanol, spin 5 min. to ensure that the DNA is pelleted.
11) Dry DNA and dissolve in TE (10 mM Tris pH8, 1 mM EDTA).
1) The precipitation of nucleic acids by spermine is very selective so this is an effective way to clean-up a DNA prep.
2) Spermine precipitation works well in 1 X TBE buffer.
3) Large genomic DNA that has been spermine precipitated takes a very long time to dissolve in the high salt. It is recommended that you not use this procedure on genomic DNA unless it is essential to eliminate inhibitors.
4) DNA in the size range of less than 50 bp and nucleotides are not efficiently precipitated with spermine. Therefore, this can be used to remove small DNA such as an oligo or fragments from the multiple cloning site in many vectors. It can also be used to remove unincorporated counts such as those leftover when DNA is labeled.