PAGE separation of 300 & 302bp fragments

[rogier]

Hi Andy,
You'll need a sequencing gel. These are usually in the 6 to 20 %
range. For a 300 bp fragnent, 8 or 10 % will do the trick.

gel: acrylamide in TBE with 7 M (!) urea.

For better resolution, it may be necessary to keep your gel
running for a while after the blue dye has run off.
I'd recommend to run your samples untill the blue dye is about
halfway, then stop the run, fill the empty lanes with the same
sample, then run till the blue dye of the second load hits the
bottom.
If you have a big gel and not too many samples, you can do 3 or 4
runtimes on one gel. This way, you're sure you have lanes where
your fragments are sufficiently far apart.

And if you still got space left on your gel, run a sequencing
reaction alongside. This is the best size marker you can get.

Good luck,

Rogier Stuger
Mol Cell Physiol, Free U of Amsterdam
E rogier AT bio.vu.nl

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