ligation/transformation: HELP ME PLEASE!!

Shin Enomoto shin at cis.umn.edu
Tue Mar 7 08:46:51 EST 1995


HARDIES at THORIN.UTHSCSA.EDU wrote:
: Heidi Moss writes:

: > [having lots of trouble cloning some HindIII fragments]
: Could it be a sequence problem?? After all, these are supposedly 
: 5'/promoter clones that have an intron and exon present, based on initial 
: analysis. Could there be weird palindromes or repeats that the bacteria 
: doesn't like?? What part to you think is throwing me off?? Also, I just 
: did an experiment where I found that the gene is also slightly induced by 
: IFNB. I am suspecting a GAS element in the promoter, perhaps. It is a 
: far-fetched hunch, but could that cause a problem?? I am ignorant. I 
: don't know what to do now.


: I hesitate to promote the idea of biological exclusion, but:
: Perfect inverted repeats do choke bacterial replication.  I vaguely
: remember that there is a host mutant that is tolerant to this problem.
: Strong promoters can cause trouble, but usually it's only for one
: orientation.  One old chestnut is that the insert permits translation
: into the lac  segment and the clones with inserts are hiding in the
: blue colonies. Heavily methylated DNA might have trouble breaking into
: a mcr+ host. Or maybe you've found some unprecedented poison
: sequence.

: Make sure the host strains haven't been switched around on you somehow.
: Your result sounds exactly like you're trying to break through the
: K, B, or P restriction system.

   How  often does restriction system interfere if the source of the cloning
involve 2 fragments derived from E. coli?  I would imagine this can only
happen if the ligation junction creates a de novo restrcition sites.
  
This restriction system interference might be more of a problem if one is
working from a PCR product. Is this encountered often?
 



More information about the Methods mailing list