HELP! RIBOPROBES.

[Klaus Salger]

sl16s at cc.usu.edu wrote:
<snip>
: All my cDNA's are in pBSKSII+ plasmid.  All cDNA's are transcribed with
: T7 polymerase  except IL-3 (T3 poly.) after linearizing with appropriate
: restriction enzymes.  Iam able to get excellent results with IL-1a, IL-6,
: TNF, and IFN-g (conditions are;  Hyb temp is 550 C, Hyb buffer contains
: 50% farmamide, Washing is high stringent(650C)- which are standerdized by
: me.) but I am getting lot of background with IL-3 and IL-2 probes.
<snip>

If pBSKSII+ contains the same MCS like pBluescript, the following might be
the problem.
Last month Andre Hamel wrote:

>Yes, Stratagene's Bluscript vectors contain sequences associated with the
>Not I site in the MCS. If one MUST use this vector for making riboprobes,
>'tis strongly recommended that the region between Sac II and Sma I be
>removed from the vector vector before use.

>This region binds STRONGLY to rRNAs ... thus, riboprobes used will give
>unacceptably high background in in situ and northerns.  Thanks to our good
>friend & consumate scientist Ingo Schroedter (U of Manitoba, Physiology
>Dept) for revealing this info to us a few years ago ... he discovered
>this the hard way (months of work) ... he's our in situ guru! :^)
><snip>

Hope this helps
  Klaus

--
Klaus Salger                phone : ++49 (0)89 5902 -502
Zoologisches Institut       FAX   :                 -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
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