Path: utzoo!utgpu!water!watmath!clyde!att!osu-cis!tut.cis.ohio-state.edu!mailrus!nrl-cmf!ames!ncar!boulder!pell
From: pell@boulder.Colorado.EDU (Anthony Pelletier)
Newsgroups: sci.bio
Subject: Re: Obtaining single-stranded linear DNA
Message-ID: <6024@sigi.Colorado.EDU>
Date: 12 May 88 17:39:46 GMT
References: <1792@aecom.YU.EDU> <5729@spool.cs.wisc.edu>
Sender: news@sigi.Colorado.EDU
Reply-To: pell@boulder.Colorado.EDU (Anthony Pelletier)
Organization: University of Colorado, Boulder
Lines: 39
Summary:Quizes? We don't need no stinking quizes!

In article <5729@spool.cs.wisc.edu> noordewi@speedy.cs.wisc.edu (Mick Noordewier) writes:
>In article <1792@aecom.YU.EDU>, diaz@aecom.YU.EDU (Dizzy Dan) writes:
>> Here's this week's biochemical puzzler (you all did well on the
>> antisense RNA trick question):
>                ^^^^^
>Really?  What was the trick?  I thought it was an honest question!
>
I thought it was an honest (albeit stupid) question also.  I figured
this dn/dx fellow as a youngish grad-student with not a whole lot
of lab experience and offered a simple way to get some genuine mutants,
rather then the questionable phenocopies one might get from antisense.
Listen, Dan, you are not dealing with a bunch of novices here.  If you think
it is your place to quiz those with little experience, please mark your
postings as such.  Do not waste the time of other scientists.  We get
plenty of legitimate "puzzles" with which to deal.

If your question was honest, I appologise.  I would be happy to offer
suggestions.  But, don't try to hide the fact that you are asking for help
by pretending it was quiz for us.

>> I am interested in obtaining tritiated (or otherwise labeled)
>> single-stranded linear DNA of uniform length (has to be at least 1knt
>> long) for exonuclease assays.
>> 
>> M13 seems ideal since the cells will spit out labeled ssDNA (circular)
>> if we cook them with label.  How do we make the DNA linear?  Do we shear
>> it, or cut it with a restriction endonuclease with ss activity
>> (expensive!) or what? The DNA does not have to be linearized in the same
>> place, but it must be linear, and cut once, or at most a few times.
>
Anyway, if the question is legitimate, consider a Polymerase Chain Reaction
on an already linear fragment.  You should get more than enough for exo assays
(after all, if one can get a single-locus RFLP map from root cells of
one hair...).  If you are set on M13 and really poor, shear it and isolate
linear.  After all, you really don't need that much.

Mazzal Tov

-tony