PAGE of short DNA fragments
rjordan at u.washington.edu
Thu Mar 16 18:03:46 EST 2000
> Does anyone know of a basic protocol or reference for running
> polyacrylamide gels for short double-stranded DNA fragments.
I've done this alot for our TRAP assays. What Adam suggests is essentially
what I do as well, except that I use 19:1 acrylamide:bis acrylamide and 0.5X
TBE. This is what's recommended by the TRAP kit manufacturer. Check out
Novex's (now Invitrogen's) web site. They sell precast TBE gels that work in
the same cells as their other (protein) PAGE gels. They work very well,
though not with Invitrogen's TRAP kit. The catalog also has a lot of info
about what gels work best to separate particular sizes, etc. With a 10%
29:1, 1x TBE gel the bromphenol blue band runs at about 35bp. With a 10%
19:1, 0.5X TBE gel the xylene cyanol band runs about 35bp. These are very
approximate. Maniatis (how ever you spell it) has a section on running
non-denaturing PAGE gels you might want to check out.
I run my gels at 150V constant voltage then stain for 10min with SYBR Gold.
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