SAGE (Serial Analysis of Gene Expression)
chrizzz at gmx.net
Sun Jun 14 14:44:34 EST 1998
I'm currently involved in establishing SAGE technique (Serial Analysis of
Gene Expression, Science 270; 484-487, 1995; Cell 88; 243-251, 1997) in our
lab and I would like to get in touch with people who have had experience or
are familiar with this method.
In my hands critical steps seem to be the ligation and subsequent
restriction of cDNA fragments bound to the magnetic particles (beads get
"sticky" during the manipulation, i.e. considerable loss of beads due to
poor dispersion) and the ligation of ditags to form concatemers.
I woul especially be interested to hear whether anyone knows of a well
procedure to quantitatively elute small fragments of DNA (20 to 30 bp) out
of polyacrylamide gel pieces. Maybe it would then be possible to
concatemerize ditags without having the preceding PCR that huge (50 to 100
reactions, as recommended by the author).
Thanks very much in advance,
e-mail: chrizzz at gmx.net
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