Plasmid sequencing: Methods to improve resolution
sullivan at gwis2.circ.gwu.edu
Sun Jul 9 13:02:00 EST 1995
S941407 at mailserv.cuhk.hk wrote:
: I would like to know how I can get better-resolution sequencing (bands
: towards the end of the gel are hardly distinguished). Moreover, are there any
: methods to increase the number of sequence obtained each time(other than
: buffer-gradient page)?
Well, if you have ~$700 to spare, you could use a sequencing apparatus
designed for 'long' gels, or , if you have *lots* of money, an automated
sequencer. But I suspect you want something cheaper....
a couple of cheap things to try: wedge gels (make them by buying
wedge-shaped spacers); these equalize the distance between bands, since
bands as the bottom (thick part) run slightly more slowly than those at
the top of the gel.
second: do two loads per sequence, spaced about two hours apart. This
gives you sequence clsoe to and far from the primer.
third: use end-labelled hot primers instead of incorporation of hot label
into the extension product. This gives equal labelling in all bands, and
is often used in cycle sequencing protocols.
hope this helps a bit -
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