Hints for low-stringency cloning

[John Watson]

In article <gdiamond.778166304 at njmsa>, gdiamond at njmsa.UMDNJ.EDU (Gill
Diamond) wrote:
> 
> 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
> UMDNJ
> "Exit 142 on the information superhighway"

Have done this, albeit a little, but what I prefer to do is keep the
foemamide constant at 50% and vary the hyb temperature --starting say, with
30 degrees or so.  Altho it probably goes without saying, the place to
start is with a lot of southerns!

John