Riboprobes, Northerns, and high background

[Mart Speek]

Gary Kucera (kucera~gt at glaxowellcome.com) wrote:
: Gil wrote:
: > 
: > Greetings,
: > 
: > I have recently been using riboprobes for radioisotopic detection of
: > mRNA species on Northern blots, and consistently get high background in
: > most lanes.  Here are the conditions:  Sodium-phosphate hyb solution
: > (Church & Gilbert); 65-68¡C hybridization; ~1 million cpms probe per ml
: > hyb solution; washes are two 15 min washes in 2XSSPE/0.5%SDS @ 65¡C,
: > followed by two 15 min washes in 0.1XSSPE/1%SDS @65¡C.

Dear Gil & Gary;

Two important aspects here which come to my mind form my own experience:

(1) Add competitor RNA (1-10 micrograms) derived from similar but 
    nonrecombinant and linearized template. This helps to reduce 
    head-hybridization of your 32P-RNA probe. I also assume you are 
    using other competitors like
    tRNA, sssDNA, polyA, etc.

(2) Reduce the hybridization time to 1-2 hours. Due to 7%SDS, 
    hybridization is frequently complete within an hour.


: You may want to gel-purify your riboprobe to collect 
: full-length copies.  The smaller riboprobe fragments could 
: be part of your problem.

(3) I don't think that this is what you need, it is time consuming and
    thus needs extra work. Cold competitor RNA should do the work
    for you!!!

Hope this helps,

Mart

> ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
> |      *                | Mart Speek, PhD                          |
> |    *   *              | Assistant Professor                      |
> |   * o o *             | 46 Vanemuise St. rm 138                  |
> |   *  -  *             | Chair of Biochemistry                    |
> |    * - *       * *    | Institute of Molecular and Cell Biology  |
> |    * - *      *. .*   | Tartu University                         |
> |    * - *      * ^ *   | Tartu, EE2400                            |
> |    * - *       *-*    | ESTONIA (Europe)                         |
> |*   * - *       *-*   *| tel/fax: 372-7-430235                    |
> |* * * - * * * * *-* * *| email: mspeek at ebc.ee                     |
> --------------------------------------------------------------------