Northern non specific ?

[Mart Speek]

Claire Fenech (c.fenech at sghms.ac.uk) wrote:
: Can anybody tell me how non specific binding of a 500bp
: riboprobe to other RNA than the target (not to the membrane
: itself) can happen...

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Yes, Yes, Yes, this seems to be a common, frequently encountered
PROBLEM for northerns. I am sure almost anyone in the field has seen
nonspecific binding to 18S and 28S ribosomal RNAs, and even so
in published papers.
This is mostly because riboprobes are "STICKY"!

I would recommend at least two strategies:

* try to use shortest hyb time, and highest hyb temp
  (2-3 hrs, ~70degC, rapid hyb cocktail cont SDS)
  Not only washing at high stringency is important!

* if probe is prepared by T3/T7 polymerase include
  a short unlabelled competitor RNA (in excess) derived by the same
  strategy (run off) from nonrec plasmid (traditionally pBS)

...this is what my experience tells me...hope this helps,


Mart

++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
|      *                | Mart Speek, PhD                          |
|    *   *              | senior scientist                         |
|   * o o *             | 23 Akadeemia tee rm 206                  |
|   *  -  *             | Center for Gene Technology               |
|    * - *       * *    | Tallinn Technical University             |
|    * - *      *. .*   | Tallinn, EE0026                          |
|    * - *      * ^ *   | ESTONIA (Europe)                         |
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