Riboprobes, Northerns, and high background

Mart Speek mspeek at tamm.eenet.ee
Tue Dec 3 01:39:33 EST 1996


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

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