sequencing from 300-500 bp

[maize at bragg.bio.purdue.edu]

In article <1er6vqINNjls at usenet.INS.CWRU.Edu>, ca566 at cleveland.Freenet.Edu (Michael Holloway) writes:
>
>In a previous article, mm6y+ at andrew.cmu.edu ("Morris F. Manolson") says:
>
>>Dear Netters,  I am sure someone must have asked this before but......
>>
>>I am trying to sequence off of one reaction 300 to 500 bps using the 
>>standard IBI or BRL 44 cm glates.  Without having to resort to ultra-long
>>plates (of which I don't have), does anyone have any suggesting on how to
>>improve the resolution of the bands in the 300 to 500 bp region.  My reactions
>>are fine and I can see bands up to 600 bp from the primer, I just can't
>>resolve them well enough to read.  I am using 6% acrylamide wedge gels and run
>>them for 24 hours at 900 Volts, this gives me nice sequence on average
>>up to 350 bp,  on a great gel I can get 400.  I remember some company trying
>>to sell an alternative matrix to acrylamide, (of course I don't remember
>>who or where) which they claimed improved resolution, does anyone
>>out there know what I am talking about?  Thanks in advance, Morrie 
>>
>
>Based on my experience (and I think the other replies to your questions
>bear me out in this) the best advice I can give you is not to try.  Bite
>the bullet, make or contract out for an oligo and prime from within your
>last bit of sequence.  Even if you do manage to get that last little bit of
>sequence out of it the quality will be extremely low.  I've been there and
>I can honestly say that I wish I hadn't gone.
>
  I just can't understand this.
  Getting out to 500 bases on wedge gels isn't that hard.  We are talking 
multiple loadings from the same reaction, aren't we?  Also this is 35-S, 
not 32-P, right?  (I use the same plates as Morrie -- I got wedge spacers from
IBI -- so I presume they are also the same...)
  Dropping down to 5% acrylamide will give a little better resolution
with the larger bands (while not making the gel impossible to handle) -- 
but even at 6% it should be easy enough to get out to 500 bases (from 
the primer) with 2 or (at most) 3 loadings.  With a good reaction (and 
some luck) I've read 700 bases from a primer.
  If there is trouble determining the order of two bases it is useful to 
load the reaction eight wide in such a way that every base 
can be seen right next to every other base (e.g., GATCGTAC).
  I don't know what kind of gel rig is being used -- but 900V seems 
unnecessarily slow to me.  I use standard IBI rigs and was able to run 
wedge gels at 1300V without heating them up too much (keeping them below
50 oC.)  I never needed to run them longer than 12 hours to see 500 bases 
out from the primer.

  In any case, I use Long Ranger gel mix now -- it performs pretty much
as advertised.  I haven't done side-by-side comparisons but I do seem to be
getting wedge gel results with normal (0.2 millimeter) spacers.  I 
generally run a 5% gel.  I use the more recessed of the universal primers
(the -40's or so) for pUC clones in my sequencing reactions.  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.  I stop the gel, fix and dry.  From an 
overnight exposure I can usually get from the cloning site to about 400 
bases out.  A third loading would probably get you out to 500.

  I get Long Ranger from:

		AT Biochem, Inc.
		30 Spring Mill Drive
		Malvern, PA  19355
		1-800-282-4626

		Cat. No. 210	125 ml (50% concentrate)
		Cat. No. 211	250 ml  

I've only ever gotten the 250 ml size and it cost $100.  (Standard gels
are 5%.)

  I haven't had trouble with it sticking to the plates any more than I do 
with standard acrylamide.  I either let the plates cool to room temp for
20 min. or so -- or IMMEDIATELY take them apart before any cooling has 
taken place.  I don't treat my plates with anything -- just wash them 
(with Alconox) until water does not bead on them.  I used to use Photo 
Flow on the short plate -- but this seemed to do nothing.

  I'd be interested to hear from anyone who often (or even occasionally)
sequences out long distances from the primer and what methods are used.  I 
actually would agree with the response to the first post above if it were 
talking about 500 to 700 bases -- just make another subclone or even buy a 
primer.  But if you are willing to run a gel 24 hours then you deserve to 
get out to 500 bases.

Phillip