another SAGE problem

Gys de Jongh GysdeJongh at compuserve.com
Mon Mar 6 17:39:59 EST 2000


"Matthias Wahl" <matthias.wahl at gsf.de> wrote in message
news:38C3E9C9.19598F4C at gsf.de...
> Dear all,
>
> I have another problem in doing SAGE. My problem lies in the
> concatenation of the ca. 26 bp ditags (after cutting the 102bp PCR
> fragment with NlaIII). Although starting with 1000ng of ditag and
> incubating for three hours, the ligation is incomplete (maximum at
> 400bp!, only ca. 10% is shifted to >1kb). I can exclude contamination
> with linkers (did already check this). Did anyone have a similar problem
> and tell me what I make wrong?
Hi,
it seems we have similar problems. We did not yet found the solution. Here are a
few thoughts : 1) we did a blunt cloning of the short constructs (to be sure to
miss nothing) , most of the short products ended in a linker. 2) there is only a
very little bit of linker needed to stop the concatenation ,1 in 400 , so you
may not notice it on a gel. 3) a tag which is dephosphorylised by the incomplete
removed cip (from the construction of the vector) is added. Than the construct
will end there. See point 2) for the amount needed. Replacing CIP by SAP , which
can be de-activated , improved things a bit. 3) a blunt tag is added. Than the
construct will end there. At this point we think that the main problem lies in
linker contamination.

--
Gys (in the same SAGE team as Bas)








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