PHAGE PURIFICATION WITH DE52

Friday, November 21, 2003

Description
PHAGE PURIFICATION WITH DE52

Procedure
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 37C.

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.

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PHAGE PURIFICATION WITH DE52