Safeguarding DNA against UV damage during isolation

Thursday, June 24, 2004

Description
DNA isolated from agarose and acrylamide gels is usually subjected to UV light in the presence of ethidium bromide resulting in crosslinks. The amount of crosslinking is proportional to the time required to identify and isolate the gel fragment. The crosslinks inhibit replication of the ligated cloned plasmid and thus can drastically reduce the number of successful transformants during the subcloning process. Simple incorporation of 1 mM guanosine into the gel and buffer system virtually eliminates crosslinking and greatly improves the success of transformation of the ligated product.

Procedure
Incorporate 1 mM Guanosine (or cytosine) into the standard running buffer (eg 1xTBE or 1xTAE). Be sure to make the gel from the modified buffer. Perform the fragment isolation as per your protocol. This protocol was published in 1996 (see BioTechniques 1996 Vol. 21 No. 5 p. 898-903)

Recipes

Supplies
Guanosine (or cytosine)

Tips

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Safeguarding DNA against UV damage during isolation