ligation/transformation: HELP ME PLEASE!!

Fri Mar 3 11:13:52 EST 1995

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

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.

Most times people claim biological exclusion, it turns out to be a
technical error.  "The technique must work because I've done it
before" ranks right up there with "I though it wasn't loaded" on the 
famous last words list. So I recommend that you try to clone some
innocuous HindIII fragment in parallel as a positive control.  Say cut
up lambda DNA with HindIII, heat kill, and directly ligate [some but
not all lambda fragments are innocuous].  If the positive control
works, then mix some of your experimental insert with it and ligate
them together.  If you suppress cloning of the control fragment by the
addition of your "cleaned" experimental fragment, then you are
carrying in some diffusible inhibitor.

If you do decide that there's biological exclusion, you might try cloning
into a phage vector.  Their hit and run life style makes them less 
vulnerable to a variety of biological problems.  

Good luck!
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC

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