ssDNA sequencing preps: NaOAc or NOT ?

Jim Graham jgraham at sunflower.bio.indiana.edu
Tue Sep 8 18:53:18 EST 1992


Maybe it's a worn thread but :

I recently have been using what was describe in a bulletin from Stratagene
as an improvement in the organic extraction procedure for cleaning up
phagemid templates. I am using it with Sequenase single stranded
sequencing. They claim that a SINGLE phenol-CIA extraction is sufficient if
the PEG phage pellet from a 1.5 ml culture supernatant is resuspended in a
400ul of  0.3 M NaOAc. Others in my lab agree, but I find that more than
half of my samples show a large grey smear instead of a ladder. Must I
return to the laborious 2 phenol/CIA, 3 CIA extraction procedure ? I have
always found that to work, but it is a real pain in the @#$#.

Thanks for your help,

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



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