cloning

[S Forsburg]

Marguerite Evans wrote:
> 
> Hello everyone,
> 
> I have a simple question to ask...how frequent/common is it for people to
> experience deletions/rearrangements when they are cloning their favourite
> gene?  I appreciate that the problem is likely to be strain-related and
> I have tried several different strains, finding my plasmid to be more
> stable in JM101 than any other.  But still, deletions occur.  We even
> jest about "vanishing genes".  Our lab often discusses possible toxicity of
> some DNA sequences to E. coli and it has been suggested to us to try directly
> transforming into yeast.
> 

It is probably more common than we realise.  I think we all have
stories about the fragment that could only be cloned in one direction,
probably due to promiscuous promoter activity on the plasmid.
Trying different coli strains can help;  especially recBC
strains like SURE or JA226;  or the very useful MC1061.   I
recall the _HAP1_ gene was very difficult to clone, probably
for this reason, and went directly into yeast without passing 
through E. coli.  However, I also recall that once passaged through
yeast it could be recovered in E. coli.  It was a long
time ago so my memory may be in error, but check out the Cell papers 
by Pfeifer and Guarente from  the late 80s.

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Susan L Forsburg PhD               forsburg at salk.edu
The Salk Institute          vox:  619-453-4100 x1341
10010 N Torrey Pines Rd     fax:  619-453-4765
La Jolla CA 92037          www:  flosun.salk.edu/~forsburg/
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