Overcoming Sequencing Secondary Structure
findley at u.washington.edu
Fri Jun 10 16:48:25 EST 1994
Here's a technical tip for those of you trying to overcome secondary
structure problems on sequencing gels. In particular, compression and
abberent migration of bands:
USB sells c7dGTP mixes in the kit, but a much more effective mix replaces
*both* dGTP *and* dATP with their c7 (7-deaza) analogs. You will need to
replace the dGTP on a per-mole basis with c7dGTP; and also replace
the dATP with c7dATP on a per-mole basis. You will need to do this in both
the Labelling and Termination mixes. You will also only be able to use a
"c-minus" labelling mix (requiring hot dCTP).
You will also need to reduce the ddGTP and ddATP concentraions by one-half.
It is sometimes necessary to follow the Termination reactions with the
Terminal Transferase/dNTP chaseto get rid of polymerase stops (bands
across all four lanes).
The above reagents do an incredibly good job at decompression. I was able
to pull twelve well-spaced bands out of a secondary structure gob that
looked like it had maybe 5 bands in it. Aberrant spacing is a pretty good
clue that there are more bands than you might guess.
Really, this stuff works great!
More information about the Methods