DD-RT-PCR Eluting Bands From Gels

[Jim Owens]

In article <2vctr2$q3k at news.iastate.edu> John Fagan, jfagan at miu.edu
writes:
>We are not having great luck eluting DD products from dried gels.

In our group we use 33P labeling.  The sequencing-type gels are exposed
to film twice so we have a template that we can cut holes in and one for
archival purposes.  The bands are cut out of dried gels that have not
been fixed.  A gel slice is placed in a 1.5ml microfuge tube with 100ul
water for 10 minutes to overnight.  The tube is heated to near boiling
for 15 minutes.  We measure the volume of liquid when transferring to a
fresh tube.  One-tenth volume of 3M NaOAc, pH 5, is added along with tRNA
to 1ug/ml.  Then we precipitate with ethanol in the usual way, and
dissolve the precipitate in 20ul water.

Hope this helps,

Jim Owens