sequencing from 300-500 bp

[maize at bragg.bio.purdue.edu]

In article <1992Nov25.123946.60 at ntet.nctr.fda.gov>, wmelchior at ntet.nctr.fda.gov writes:
>In response to an earlier query, Phillip, wrote in part:
>
>>I run the 
>> xylene cyanole of the 1st loading just off the gel (or sometimes wait an
>> extra 1/2 hour) and then load a second time.  The 2nd rxn. are run until the
>> bromphenol blue is just off.
>
>Phillip-
>
>Did you by any chance get these backwards?  I don't usually go as far as 
>you (usually slightly > 300 bp), but I run my first loading till the
>bromophenol blue (the slower dye) is off, and the second loading for about
>15 minutes after the xylene cyanol is out.  (The last 15 minutes gets rid
>of sequences between the primer and the region of interest.  I wouldn't go
>quite as long if I wanted to read closer to the primer.)  If I were trying 
>to read longer sequences, I'd want both loadings to go longer, but I think 
>I'd have to increase the time for the first loading more than the time for 
>the second loading.
>________________________________________________________________________________
>The opinions stated are mine, not those of NCTR or its sponsoring organizations.
>
>Bill Melchior                                ||     OMNISCIENCE
>National Center for Toxicological Research   ||    Knowing what
>Jefferson, AR  72079                         ||    thou knowest not
>(501) 543-7206                               ||    is in a sense
>                                             ||    omniscience.
>WMELCHIOR at NTET.NCTR.FDA.GOV                  ||       from Grooks, Piet Hein

  No xylene cyanol is the slower dye.  It runs at 170-180 (on a 5% Long
Ranger gel.)  Bromphenol blue runs at 40 bp.  Recent gels have convinced
me that it's better to start the second loading just as XC is going off
or a little earlier.  Otherwise I end up getting some sketchy sequence in 
the middle.


_______________________________________________________________________
Phillip SanMiguel               Purdue
maize at bilbo.bio.purdue.edu       Institute for
                                  Molecular    "We'll do anything
  Bennetzen Lab                    Plant          for funding."
                                    Science
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