DD-RT-PCR Eluting Bands From Gels
jow at helix.nih.gov
Wed Jul 6 15:01:39 EST 1994
In article <2vctr2$q3k at news.iastate.edu> John Fagan, jfagan at miu.edu
>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,
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