I need some help from those of you who were able
to get good reads (>100 bases) from Lunatic
template using 377 XL sequencer. One of my clients
has *finally* found their long sought after DNA.
Unfortunately, it is GC rich, and my protocol is
not getting past about 70 bases. I didn't fare
much better on Lunatic. How can I get through this
GC area? Here is what I am doing:
3ul Big Dye dGTP
3 ul Mg buffer
300 ng template
3.2 pmol primer
(10 ul final)
94 deg. 5 min
30 cycles of
94 deg. 10 s
55 deg. 5 s
65 deg. 2 min
I am going to try with dRhodamine using a full
20ul reaction. Does anyone have any other advice?
Thanks so much in advance-
~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Sheryl Christofferson
OMRF DNA Sequencing Facility
Oklahoma City, OK
sherylc@omrf.ouhsc.edu