sect22
1. Grow fresh o/n of appropriatre cells (eg. KW251.
2. To 10ml LMM in a 100ml flask add 0.2ml o/n and a single plaque.
3. Let stand for 10min. then shake for 3-4hr until lysis has occurred. Add 0.5ml CHCl3 and shake for a further 15min.
3. Spin to remove debris and use 0.5ml of lysate for DNA prep.
Prepare slurry of DE52:
12g DE52 plus 36ml 50mM HCL (conc. is 8.7M) make about 28ml final slurry.
Add conc. NaOH to pH 6.8.
Let settle and remove s/n
Add several volumes of LMM and let settle.
Remove s/n and repeat 3X
4. Add 0.5ml lysate to 0.5ml of well mixed slurry. Shake X20 and spin out DE52.
5. To s/n add:
25ul 10% SDS
10ul 0.5M EDTA
Let stand for 5 min. at 37¡C.
6. Extract 2X with Phenol:CHCl3, 1X with CHCl3
7. Add 1/10 vol. 3M NaOAc and 2.5 vol ethanol.
8. Spin 15min. Final yield should be about 5ug.