Question on Plasmid Cloning
chrisb at hgu.mrc.ac.uk
Tue Aug 24 02:07:46 EST 1999
John Dixon (jpcd100 at mole.bio.cam.ac.uk) wrote:
: > Student <mucineer at iname.com> wrote (snipped):
: > >> is there anything in the process that prevent different molecule of the
: > >> insert from "self-ligating" to form species such as
: > >> HindIII-------BamHI--------HindIII or BamHI--------HindIII------BamHI?
: > The answer is that NOTHING prevents this happening in the ligation:
: >What you can get, however, is a recombinant in which a mutational event
: >has disrupted the palindrome(s) in some way so as to restore viability.
: >I have never seen this happen despite giving ample opportunity.
: I have been working with some plasmids that contain two HSVtk genes
: which are in the opposite orientation to each other (for eventual
: negative selection in targeting constructs - derived from lambda KO
: (Nehls et al., Biotechniques 1994)). The plasmids dont yield as much
: DNA as some but when an insert is placed between the TK cassettes, they
: grow as normal.
: I was assuming that the bugs (XL10 - recA- etc) are not entirely happy
: growing the "empty" vectors where the TK cassettes are back to back
: with only 60-80bps of polylinker between, but can replicate the repeats
: no problem when the are interrupted by the insert of >5kb.
: From what Chris has said though, should I be worried that one (or both)
: of the TK genes have picked up enough mutations for them to be stably
: replicated but no longer functional when returned to eukaryotic cells?
: Or do the palindromes have to be exactly back to back for the problems
: to start and mine grow OK (but grudgingly) because there is some
: interruption (60bpS) and grow fine with a big interruption.
Got it in one. The palindromes have to be exactly head to head to be
lethal. I believe even quite a small gap of a few non-palindromic
bases will relieve the effect: 60 bp should be enough and anything over
5 kb would certainly be sufficient. Search Medline on palindrom* and
Leach DRF (author) for many appropriate references. As for mutations
arising, that is a possibility but I think you're more likely to get
deletions or insertions that would be immediately obvious. A transient
tranfection assaying for TK activity would put your mind at rest more
Chris Boyd | from (but not \ MRC Human Genetics Unit
Christopher.Boyd at hgu.mrc.ac.uk | much longer) / Crewe Rd, Edinburgh
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd EH4 2XU, SCOTLAND
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