Greetings:
I use betaine, but with the dRhodamine chemistry on a 377. I have tried
several ways and this seems to work the best.
8 ul Rhodamine (not less)
4 ul betaine
2 ul water
4 ul template (100 ng/ul)
2 ul primer (3.2 pmol/ul)
PCR profile:
95 deg. 5 min, then
94 deg. 40 sec
50 deg. 15 sec
60 deg. 4 min (not less)
cycle 60 times (not less)
I have been able to sequence a template 91% GC. I haven't tried this using Big
Dyes yet.
Good luck!
Sheryl Christofferson
OMRF DNA Sequencing Facility
Oklahoma City, OK
sherylc@omrf.ouhsc.edu
www.omrf.ouhsc.edu/OMRF/Core/DNASEQ/gc_gt70.html
Margarita Almeida wrote:
> Hello. I am having difficulty sequencing GC rich templates. I have a 373
> with XL upgrade and use Big Terminator chemistry. I have heard of a product
> called Betaine. Has anyone used it or does anyone have any other
> suggestions?
>
> Thanks,
> Margarita Almeida
> University of Miami School of Medicine
> Department of Biochemistry and Molecular Biology
> DNA Core Facility
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