Sequencing. How Far?

John Nash nash at nrcbsa.bio.nrc.ca
Mon Nov 15 09:29:50 EST 1993


In article <2c7313$62s at nuscc.nus.sg>,
Chia Jin Ngee <mcblab47 at leonis.nus.sg> wrote:
>
>Hi to all!
>
>Yes! It is sequencing again. Must be boring you guys in cyberspace. I 
>have this problem.

Naaah, this is bread and butter stuff for you and me, it can't be boring. ;-)

>
>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? 
>

Seriously, if template purity, etc. are not a problem, you may want to
give it up, and make new primers closer to where you lose resolution.
We've found that it is quicker to make the primers than to try and
resolve a problem like this.  Of course, I'm assuming the luxury of inhouse
primer synthesis.

If not, I did try running a slow (1000 - 1200 V) gel (at least 8 hr) a
couple of times, and got nice resolution.  This was a few years ago at
another time and place, so I don't have the record handy... bear with
my memory.

Remember when those LONG range Klenow protocols came out a few years
ago?  Well I tried them...  what I did was sequence some M13 template
with some primer, and loaded the gel every few hours.  The triple
loaded sample was still clearly readable.  With Sequenase, I bet it
would be just as nice.  This worked MUCH better with ssDNA than with
dsDNA, though.  The pain was nursing a gel for a few hours (I changed
the tank buffers for the third load) and generally using Klenow to
sequence!

>Any tricks, tips and magic involved in this? I hope it is not those "your
>mileage may vary" kind of scenarios. Thanks in advance. 

Like I said, making new primers was less of a hassle.

-- 
John Nash                           (nash at nrcbsa.bio.nrc.ca)
Institute for Biological Sciences,  National Research Council of Canada,
                 Yet another Aussie-in-exile ;-)
      *** Disclaimer:  All opinions are mine, not NRC's! ***



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