Amberg Lab ,Upstate Medical University

http://www.upstate.edu/biochem/amberg/protocols/seq_temp.html

Notes on DNA : The cleaner the better.If mini DNA use phenol extracted and use the entire mini prep for each reaction.For best results use Qiagen maxi prep DNA that has been phenol and chloroform extracted then precipitated through ethanol and speed vac'd to dryness for 30 min.(get rid of the acetate).

1.In an eppy tube combine 6μg DNA plus 6μl 1M NaOH (fresh from fairly fresh stock)and take the volume to 24μl with H₂O.

2.Denature at 85℃ for 5 min.and plunge into a ice water bath.Add 3μl cold ammonium acetate (2M pH 4.5)and 70μl 100% EtOH.Store at -20℃ for 1-2 hrs.

3.Spin down DNA 10 min at high speed.Wash pellet with 70% EtOH,repeat a short spin.Pour off EtOH and dry in a speed vac for 30 min.This drying is important,any residual ammonium acetate will affect the gel.