Help! Unexplained deletion in gene

Robert Preston rapr at med.pitt.edu
Tue Apr 4 16:41:05 EST 1995


In article <Pine.SOL.3.91.950403173923.1320A-100000 at chip>, Robert Brushia
<ez017144 at peseta.ucdavis.edu> wrote:
> I don't really have an explanation for your problem but I would suggest 
> checking your RNA, perhaps it has the deletion already in it.

Makes no sense, since PCR from the RNA gives a full-size product.     

>  On 24 Mar 1995, Ramkumar Lachumanan wrote:
> 
> > Dear Netters, 
> > I am trying to clone and sequence a known toxin gene (180bp) from snake and 
> > been facing with the problem of deletion (45bp) in the gene . The 
> > deletion occurs at the same position and there is a frame shift 
> > where the deletion occurs everytime. 

Possibly a toxic (to E.coli) gene.  
1. Don't bother cloning: use PCR for everything.
2. try to clone with a different vector to avoid possible promoter activity  
   with the current vector.
3. Grow transformants at 30C instead of 37C. (this trick eliminates
   deletions in HIV cloning work here...I don't know who discovered the
   anti-deletion effect of the lower temperature, or whether that is HIV-
   specific or not)

-- 
Robert Preston                                    rapr at med.pitt.edu
University of Pittsburgh                        Pittsburgh PA 15261



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