Size-selection of cDNA: method?

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Wed May 16 08:38:19 EST 2001


When I made a cDNA library last year, I used a spin column product from
Pharmacia (no affilitation) called SizeSep 400 (cat# 27-5105-01).  It was
very effective in my hands (although I had to make a few minor
modifications to their protocol-- all having to do with the mechanics of
spinning the column).  A few things relevant to you-- you probably won't be
able to recover the less than 400 bp fraction.  Also, for the volumes, I
don't have the protocol handy, but I had my cDNA in 100 ul and recovered
about 70 ul in which >50% of the cDNA was present.  For subsequent
ligation, I had to precipitate the cDNA, but that was not a big deal, and I
recovered the vast majority of the cDNA.

Hope this helps.  If you decide to use this, I can send you information
about the spin set up modifications I made.

Mike



>Greetings!
>
>I have a cDNA library that has rather too many small inserts. I still have
>some of my cDNA substrate I used for the ligaiton, but not a lot of it
>(ca. 100ng). Ideally I'd like to separate out the short cDNA molecules -
>say retaining everything over 400 bp. I could obviously do this via gel
>extraction, but I'd rather not go that route for fear of ending up with
>too dilute a product.
>
>So.....what I'm after is a method (or suggestion of commercial
>size-exclusion columns) for removing DNA <400 bp from a small volume
>(currently I'm in 30 ul, and ideally wouldn't go up to more than 50 ul).
>Ideally I'd be able to get the <400 bp stuff back as well, but that's of
>lesser importance.
>
>Any suggestions?
>
>Cheers in advance
>
>Chris
>
>--
>------------------------------------------------------------------------------
>Dr. Chris Coward                       | Tel: +44 1223 333976
>University of Cambridge                | Fax: +44 1223 333992
>Department of Genetics                 | E-mail: cc122 at mole.bio.cam.ac.uk
>Downing Site, Cambridge                |
>CB2 3EH                                |
>England                                |
>------------------------------------------------------------------------------


Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

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