ssDNA sequencing preps: NaOAc or NOT ?

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Tue Sep 8 21:14:47 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.................


I have always used one phenol extraction then one IAC extraction, never
twice with excellent results.  BUT the major problem is with the removal of
the PEG.  ALWAYS aspirate, then briefly respin and re-aspirate to dryness. 
The PEG causes major problems with sequencing.

Hope that this helps.

Cheers, Klaus. 
--------------------------------------------------------------------------
Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"Don't try and fix it if it ain't broke".
--------------------------------------------------------------------------




More information about the Methods mailing list