sequencing from 300-500 bp
maize at bragg.bio.purdue.edu
maize at bragg.bio.purdue.edu
Mon Nov 23 19:34:42 EST 1992
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
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
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.
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