TA Cloning questions...

Chris Boyd chrisb at hgu.mrc.ac.uk
Thu Aug 13 06:19:49 EST 1998


Koichi Kunitake (kunitake at salk.edu) wrote:
:    I was reading the Promega TA cloning guide to its pGEM system, and it says:

<snip>

: Also, I was wondering it it would be better to use E Coli DNA ligase
: instead of T4 DNA ligase in the ligation, since the E Coli DNA ligase
: would be unable to close up the blunt ends, but hopefully would be able to
: covalently join T/A overhangs. (1 base counts as "cohesive", right?) Has
: anyone tried this?

Bad idea. E. coli DNA ligase does not work intermolecularly at all
under standard conditions.  See

1. Hayashi, K. I., Nakazawa, M., Ishizaki, Y., Hiraoka, N. and Obayashi,
   A. (1985) `Stimulation of intermolecular ligation with Escherichia coli DNA
   ligase by high concentrations of monovalent cations in polyethylene
   glycol solutions.' Nucleic Acids Res., 13, 7979-7992.


Best wishes,
-- 
Chris Boyd                      | from, but not \ MRC Human Genetics Unit,
Christopher.Boyd at hgu.mrc.ac.uk  | on behalf of  /  Western General Hospital,
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd \   Edinburgh, EH4 2XU, SCOTLAND



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