ALKALI HYDROLYSIS OF BAC DNA BEFORE RANDOM-PRIMER LABELLING

Breaking (shearing) DNA by Alkali Hydrolysis

Suitable for BAC clones before labelling using  random-primer (random priming, oligolabeling) labeling (which is best and most random with shorter fragments than the full-length BACs; autoclaving of syringing the DNA requires too much volume).

300 ng DNA + 6.5 ul 4M NaOH made up to 155 ul, final NaOH 200 mM

Mix and incubate room temperature 20min

Add 65 ul 5M ammonium acetate, 550 ul ice cold 100% ethanol (total volume 770ul)

Allow to precipitate (e.g. 2 hr), centrifuge as usual and redissolve are required.

Trude Schwarzacher

September 2004.