Size fractionating before making cDNA library

Hiranya Roychowdhury hroychow at NMSU.EDU
Fri Oct 24 18:12:12 EST 1997


At 04:29 PM 10/24/97 -0500, Timur Yarovinsky wrote:
>This is a novice's quiestion.
>
>I am trying to make a cDNA library with Lambda ZAP II vector. I need to
>size fractionate cDNA, which was ligated to adaptors, before ligating to
>vector arms. I did not label the cDNA and have less than 300 ng of cDNA,
>that's why I cannot monitor fractions collected from Sepharose column
>neither with counter nor on a gel. I have a stupid idea to add some
>markers (Lambda or Phix 174 digests) to my sample to monitor the fractions
>on agarose and/or polyacrylamide gel.
>
>So, your critique is wellcome.
>
>Timur Yarovinsky, Ph.D., M.D.
>Oakdale Research Park, B141 MTF
>2501 Crosspark Road
>Coralville IA 52241-8802
>
>tel.    (319) 335-4608
>fax     (319) 335-4347
>e-mail: timur-yarovinsky at uiowa.edu
>


This is what I think:  Since it was not size-fractionated before ligating,
your suggested approach is worth the try. Run the sample out on an agarose
gel with the appropriate markers and then collect fractions on
DEAE-Cellulose membrane(s) by "tombstoning". For  example, if you are just
trying to collect everything above 0.5kb, simply insert the membrane at that
position and electrophorese everything above that size onto it.




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