ligation/transformation: HELP ME PLEASE!!
lyons at cyclid.demon.co.uk
Wed Mar 8 06:44:01 EST 1995
In article <D52oz7.5n5 at news.cis.umn.edu>, shin at cis.umn.edu (Shin Enomoto) wrote:
> 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?
I have used an E.coli strain called CES201 (in Maniatis) which is tolerant
to repeat sequences as it's recF pathway is disrupted. Fragments
containing lots of repeats which were being spliced out in other strains
were stable in this strain. Might be worth a try.
merry at cyclid.demon.co.uk
\\ // Alan Lyons \\ //
\\ // Email lyons at cyclid.demon.co.uk \\ //
|| Tel. 0753 534655 ||
|| Molecular Biology Dept. ||
|| Celltech Therapeutics Ltd. ||
More information about the Methods