DNA from P22 heads (old method).
modified by Eric Kofoid April 27, 1998
Innoculate 25 ml LB with a 5 mL overnight culture
Add, using gloves and exceeding care in the hood, 2 µg/mL mitomycin C
Grow with shaking overnight at 37¡.
Add 3 mL CHCl3, shake vigorously for 3'.
Spin out cell debris 8K for 15 min.
Spin down phage 17K for 2hrs.
Resuspend phage in 0.5ml buffered saline.
Extract with1 volume chloroform, keep aqueous phase.
Add DNase and RNase to a final conc. of 1 and 10 µg/ml, respectively.
30 min. at 37¡ C.
Extract with buffered phenol until the interface is clean.
Extract with 50:48:2 phenol:chloroform:isoamyl alcohol.
Add 0.3 volumes 7.5 M NH4OAc.
Precipitate with 0.6 volumes isopropanol.
15' full speed in microfuge.
Wash pellet in 70% EtOH, dry briefly, resuspend in 100 µL H2O.
Last Update: Thursday, 19-Jun-2014 11:59:46 PDT
This page has been viewed 302 times.
eckofoid at ucdavis.edu