DD-RT-PCR Eluting Bands From Gels

DD-RT-PCR Eluting Bands From Gels

I am using the procedure of Weaver, et al. BioTechniques 16:226-? 1994.

They cut bands from dried, silver stained gels and just put them into PCR reactions. No prior hydration. Seems to work fine for me.

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.