IUBio

Sequence problems

Paul Rasmussen paul at ib3.gmu.edu
Tue Mar 23 12:04:14 EST 1999


I have two pieces of advice.  1st. Perkin Elmer has a dGTP BigDye cycle
sequencing kit which works very well if you can get your hands on an ABI
310 or 377.  Actually you could check if the kit you're using substitutes
dInosineTP for dGTP.  Some kits do this to help smooth mobility down gels
from templates with potential secondary structures/ interactions.  dITP
doesn't really sequence through high G content regions well though
(especially poly G strings).  So basically
    2nd. OPTIMIZE YOUR REACTION  Sequencing, like PCR should always be
done using the highest possible annealing temp to prevent secondary
structure in your template.  Good luck and email me if you have any other
problems or questions.
Paul

amherrera at CIGB.EDU.CU wrote:

>     I'm trying to sequence a synthetic GC rich gene. I'm using Tdt
>     and the result is that I'm just able to read about 40 bases of the
>     central region.
>     Could you please tell me any possible solution for this trouble?
>     Thanks in advanced
>     Antonieta

--
Paul Rasmussen
Research Associate
Institute for Biosciences, Bioinformatics, and Biotechnology
George Mason University, Manassas VA
Phone: (703) 993-8463     Fax: (703) 993-8460
paul at REMOVE TO REPLY.ib3.gmu.edu              www.ib3.gmu.edu

"Whenever I watch TV and see those poor starving kids all over the world,
I can't help but cry. I mean I'd love to be skinny like that but not with
all those flies and death and stuff."
-Mariah Carey, pop singer





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