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Searching for advice

Marc Marc
Tue May 13 09:13:11 EST 1997


Maria,

        The unpredictable results you describe may well be due to trouble 
in
the denaturation step. The AutoRead kit protocols involve a NaOH
denaturation/HCl neutralization step. If you can robustly get 700-800s 
with
the M13 ss control (no denaturation) but have trouble on ds templates, 
this
is likely the cause. Most common problems leading to this involve using
something other than a P2 pipettor (larger ones are not reliable for this
step), getting small droplets of the solutions on the outside of the 
pipette
tip (so that the wrong volume is being added to the reaction), or the NaOH
or HCl aliquots are too old. Careful attention to this step is crucial.

        Another common trouble is failure to use enough template, or using
template that is not sufficiently purified. If a "good" template
consistently gives good results but other templates don't, uneven
purification/concentration could be at the root of your result 
variability.

        You might try using a cycle sequencing kit as an alternative if 
this
does not help. Generally, cycle sequencing requires much less template and
avoids the need for chemical denaturation steps. Most commonly available
kits for automated cycle sequencing with dye primers work well on our 
system.

        If you need further help feel free to email me, but the local
specialist who installed your system can probably provide the quickest
response, and he or she will know more about your specific case.

        Sincerely,

        Marc Goldstein
\\/\\/\\/\\/\\/\\/\\/\\/
Marc A. Goldstein, Ph.D.
Applications Specialist
Molecular Systems
Pharmacia Biotech, Inc.
2202 N. Bartlett Ave.
Milwaukee, WI 53202
U.S. Voice Mail: 1-800-526-3593, 1, 5057
-----------------------------------------------------------------------
At 11:20 AM 5/12/97 -0700, you wrote:
>Dear all:
>I'm getting started in DNA automated sequencing. I'm working in a
>Research Institute from Buenos Aires, Argentina. We've got an Alf
>Express from Pharmacia. We're using the dye-primer chemistry with T7
>polimerase (Autoread kit, Pharmacia) and Long Ranger gels (5.5%) for
>sequencing double strand DNA. I'm not very satisfied with the results
>that seem to be quite impredictable (althoug following scrupulously =
>the
>recommended protocols and care of plates,etc). I'd like to be in touch
>with others Alf's users (I'm pretty sure ours is the only one in
>Argentina) and moreover I'd like to know if any group in London is
>working with an Alf (I'll probably be there on October-November '97).
>Any help and advice are very welcome.
>
>You can mail me directly to:
>
>ruben at saveas.com.ar
>
>Thanks in advance
>Maria
>
>

\\/\\/\\/\\/\\/\\/\\/\\/
Marc A. Goldstein, Ph.D.
Applications Specialist
Molecular Systems
Pharmacia Biotech, Inc.
2202 N. Bartlett Ave.
Milwaukee, WI 53202
U.S. Voice Mail: 1-800-526-3593, 1, 5057
Canadian Voice Mail: 1-800-992-5023, 5057
magoldst at ix.netcom.com




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