Ligation

[Tim Fitzwater]

Desphosphorylating the vector for several hours with CIP is extremely risky and contaminants in the prep have probably damaged the ends of the vector.  CIP and BAP should be used precisely (unit definitions and recommended units/ug of DNA vary by supplier).  They are usually used at high temperatures because that inhibits (but does not eliminate) the contaminating exonclease activity.  When using these enzymes, I dilute the CIP/BAP as recommended for the number of ends (sticky or blunt) and react at the temp and time recommended.  I then clean the DNA thoroughly and repeat the CIP/BAP one more time because the reaction is self-inhibiting and does not go to 100% the first time.  SAP is much more forgiving and can be used simultaneously with the restriction enzymes.