Single-strand rescue of phagemids

the End jgraham at bronze.ucs.indiana.edu
Fri Oct 23 14:09:39 EST 1992


Check the Focus article from last year. They found that you can be quite 
sloppy and get good yields (eg. start from a resuspended colony or stationary
batch, throw in some helper and let it shake overnight). You don't need 
Kan, and I think it decreases the ratio of phagemid strands to helper.
I use the TB (buffered rich) broth and grow a minimum of 14 hours on a 
tube roller for max aeration. When growing deoxyuridine containg DNA in
CJ236, I grow them for 16 to 18 hours. These extra hours seem to increase
yields substantially. I have inserts less than 1 Kb in Bluescript, so that 
may be why I don'tsee rearangement with longer growth. Percipitate both
phage and DNA with amonium acetate salts to improve polymerization on the
templates.

As far as mutiple subculturings and yield, I cannot comment as I can't 
imagine why someone would need to grow the phages every day or two for a month 
(ie. I start overnight from frozen stocks when I need to make some ssDNA).


Hope this helps,

Jim
J. Graham
Biology and Chemistry Depts.
Indiana University -Bloomington
.................."Il n'y a pas sciences apliquees, mais il y a 
...................des applications de la science". L. Pasteur................



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