BMCCARTHY.LUDWIG@BIONET-20.BIO.NET (Erwin Ludwig) (04/14/89)
Tacking on a restriction site on your pcr oligos helps alot, such as using a 24mr with a Hind III or Eco RI site in it.It shouldn't make a difference if the 5 prime end is floating in the breeze. Another more obvious solution is kinasing the blunt end fragment, since pcr oligos do not have 5 prime phosphates they will not insert well. Add a pinch of 32P gamma label when end labeling and run it on a gel prior to ligation, the 32p will not interfere with ligation. It wouldn't hurt to phosphatase your ve ctor to. Erwin -------