In response to Margarita Almeida
Our approach to high GC templates follows what Laura Livingstone of
UNC Chapel Hill suggested.
Use standard amount of DNA.
Alter cycling conditions. Below assumes use of 200 ul thin wall
tubes in a 9600, 9700 or 2400.
95 C for 5-8 minutes
one cycle of
98 C for 45 Sec
50 C for 10-15 sec
60 C for 4 Minutes
cycles 2-30
98 C for 15 Sec
50 C for 10-15 sec
60 C for 4 minutes
use 2 ul of 50% DMSO
When possible design custom primers with Tm over 60 C and then do 2
cycle sequencing with annealing and extension at 60 C.
We found no improvement with Betaine.
Hal
>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
Harold G. Hills, Ph.D. DNA Sequencing Specialist 515 294-9585
1184 Molecular Biology Building FAX 515 294-1597
Iowa State University hhills@iastate.edu
Ames, IA 50011-3260
http://www.biotech.iastate.edu/Facilities/DSSF/ABRF For ABRF 97 DNA
sequencing tutorial.
http://www.biotech.iastate.edu/Facilities/DSSF For info about facility.
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