Preferential insert orientation

Paul N Hengen pnh at fcs260c2.ncifcrf.gov
Thu Dec 30 13:41:29 EST 1993


In article <2fv12l$af5 at polaris.unm.edu> bhjelle at unm.edu

Brian writes:

| I certainly have noticed this problem while "TA" cloning, another seemingly
| symmetric cloning strategy. I wanted clones in a particular orientation so I
| could use the neighboring polylinker sites for subcloning. In one example, 17
| out of 17 clones went the wrong way!  Other inserts that shared the same 3' 
| end (of the ORF) refused to go in the correct orientation as well.

A few years back, I tried to force clone into restriction sites located at one
end of a large polylinker sequence and found that some of the restriction sites
within the multple site region were missing after the clone was constructed
and characterized. I thought this to be a result of many palindromes linked
very closely together, but never really went after the Nobel Prize, that is,
figuring out exactly what caused it. I moved on with the designed experiments
on the agenda instead.  I still wonder if anyone else has seen this. My thought
is that you may get both orientations of your fragment in a vector with a
single restriction site, and that a huge multiple cloning site nearby could be
giving you difficulties. On the other hand, I was working with a potentially
lethal gene and the deletions could have allowed for lowered expression. The
most obvious deletions would have been within the gene itself and I never saw
any of these, so I've always scratched my head over this one.

-Paul.

*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
*******************************************************************************



More information about the Methods mailing list