primer annealing for cloning

Ava spirit-images at
Sat Jun 10 21:21:54 EST 2006

I tried a procedure were 2 different primer sets were boiled for 5 min. in STE buffer and then allowed to cool to RT.  One set of primers were at 1mm and the other at 200 um.  For ligation they were used at 5 ul, 1 ul and 0.1 ul.  The plasmid was cut with restriction enzymes and purified.  The ligation was done in a total mix of 20 ul, with the above insert concentrations and 1 ul out of the 30 ul purified plasmid, 2 ul ligase buffer and 1 ul ligase.  Ligation was incubated overnight in a cold water bath and transformation done as usual.  

Colonies were screened, and although the inserts should have gone into the plasmid without a problem, what happened is that the cut restriction site overhang of the vector became blunted, and the wrong sequence was cloned in.  I am at a loss as to how this could have happened.  The vector was cut overnight with HindIII and NheI.

Does anyone have a clue as to why the procedure did not work correctly?

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