SS Phagemid gradients, KI or CsCl?

Pierre Jelenc pcj1 at cunixf.cc.columbia.edu
Thu Jul 23 13:40:12 EST 1992


In article <1992Jul23.025622.9263 at menudo.uh.edu> bchs1b at Elroy.UH.EDU writes:
>In article <1992Jul21.154049.25431 at usenet.ins.cwru.edu>, djt2 at po.CWRU.Edu (Dennis J. Templeton) writes:
>>
>>We're trying to purify a mg or so of phagemid DNA, and need it to be free
>>from contaminating double stranded DNA, such as might come from broken
>>cells.  The protocol (sketchy) we're following suggests using EtBr/KI
>>gradients for this, and collecting the lower band (SS) and discarding the
>>upper band.  Our first go at this was a bust, as we lost almost all of the

>
>Instead of running gradients, why don't you treat the lysate with DNAse,
>which will not get into the phage heads, then PEG ppt your phage (twice
>if you want to be paranoid) phenol extract and etoh ppt. Should be
>free of ds DNA.
>
It does not quite work. In our hands, there is always some ds DNA left.

Pierre


Pierre Jelenc                        pcj1 at cunixf.cc.columbia.edu 
                                    Columbia University, New York



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