isolating 197bp fragment

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Wed Nov 22 11:31:18 EST 2000


>I need to isolate a 197bp dsDNA fragment in sufficient quantities for
>making a radioactive probe. I have the fragment subcloned in a plasmid
>and can easily isolate it from the plasmid with an EcoR I digest.
>

For random primed labeling of fragments, you can isolate a piece of low
melt agarose containing the fragment and use that appropriately diluted
directly in the labeling reaction.  With small fragments like yours, just
make sure to start with enough DNA such that following dilution you can
include enough DNA in the reaction.

The usual dilution I use for a 1% gel is 3 ul of water for each mg. of gel.
Thus for a 100 mg gel slice, melt the fragment in 300 ul water.

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax


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