Also important is the gel buffer used: avoid TBE for gel extractions, as Boric acid at that molarity can bind DNA and inhibit downstream applications such as spin columns.
I would recommend looking up Sodium Borate as a gel buffer. It permits higher voltages without heating, which saves time, it permits downstream applications and gel extractions, and it offers good resolution. Also, it's about 35 times cheaper. If you want to try it out, make up 1x Sodium Borate buffer with 1.907g of disodium Borate decahydrate in 1l of distilled water, optionally filter sterilise. Also see the faq on fasterbettermedia.com. All the best, Cathal On Feb 5, 2010 5:31 AM, "Adam ." <[email protected]> wrote: The binding efficiency of spin columns depends on pH and salt concentration. Generally the binding buffer you dissolve the gel slice in should have sufficient salt and pH. The columns work really badly to bind DNA if the pH is greater than 7.5. Some protocols suggest that you spike in a little 3 M sodium acetate pH 5 before running it on the column. Elution off of the column is also pH and salt dependent. You want a higher pH buffer to elute in. There are also binding issues with the size of the DNA fragment. There is an optimal range of size over which the columns are supposed to work. Generally, most of this stuff is found in the back of the manual for your spin column. You might get some insight by calling up technical support. In my experience, even though the manufacturer claims you get 99% recovery, you rarely do and are lucky if you get 50-70%. Adam On Thu, Feb 4, 2010 at 7:41 PM, Adroit <[email protected]> wrote: > Dear Bionetters, > > > ... _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
