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).
- In an eppy tube combine 6ug DNA plus 6ul 1M NaOH (fresh from fairly fresh stock) and take the volume to 24ul with H2O.
- Denature at 85¡C for 5 min. and plunge into a ice water bath. Add 3ul cold ammonium acetate (2M pH 4.5) and 70ul 100% EtOH. Store at -20¡C for 1-2 hrs.
- 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.
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