Hints for low-stringency cloning

[Gill Diamond]

tshin at fas.harvard.edu (Tae Bum Shin) writes:

>Hi there;
>  We're currently trying to clone a homologue of a gene by using 
>the original cloned gene as a template for a random-primed probe.  Does 
>anyone have any hints or suggestions on hybridization/washing 
>conditions?  

>Thanks;

>T.B. Shin
>tshin at fas.harvard.edu


TB, I've done this alot, and it's really quite a game.  Depending on the size
of the probe (I sometimes use oligos) and the extent of homology, I'll vary
the hyb or wash conditions.  Try to hyb at 10-20% formamide, and start with
a relatively low stringency wash, like 2X SSC at 55 degrees -- you can always
go up.  Small changes in temperature of the wash can make a big difference,
so  I usually stick with a single concentration of SSC and slowly increase
the temp.  Good luck.  -- 
Gill Diamond, Ph.D.			Internet: gdiamond at umdnj.edu
Department of Anatomy, Cell Biolgy	Voice: (201) 982-3324
   and Injury Sciences			Fax:  (201) 982-7489
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