Sequencing. How Far?

[Martin Kennedy]

In article <2c7313$62s at nuscc.nus.sg>, mcblab47 at leonis.nus.sg (Chia Jin Ngee) writes:


> I've tried dsDNA and ssDNA sequencing and I can't seem to read beyond 300
> bp. I use Sequenase with 1X labelling mix and I rarely get beyond 300 bp. 
> With 5X labelling mix, I know I get way beyond 300 but can't seem to
> resolve the ladder. I've run a gel for 6 hrs and I just get a blur of
> ladders. I use a BioRad 38X50 cm apparatus and I run a 0.4 mm 6% gel at
> 100 - 120 W to maintain 50 deg C. What could be the problem here? Is it
> the gel, rxn or template? 

Hi,

We use a more or less identical set-up for ds-sequencing, except we usually use
the narrower BIORAD Sequigen set-up.  We also tend to get less than 300bp with
this gel system; we use ready-made gel solutions (home-made, but kept at 4
degrees for up to several months), don't degas the gels and generally take
quite a few shortcuts.  The net result of all this is that I never count on
being able to read more than 300bp, and prefer to either use another clone as a
template, or get another primer made.  My experience of long runs is that they
can give you an extra 50bp or so, but without a lot of fiddling we don't get
any more than that.  With ss templates, we could on a good day, get 400bp, but
I'd hardly call this a routine event.  This doesn't answer your question, but
maybe it'll make you feel better:-)


Cheers,

Martin

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