Ligation problems on agarose-isolated fragments

[Riekeltje Koedood]

Mark R. Miglarese (miglaresemr at pfizer.com) wrote:
: 

We don't isolate the DNA from Agarose to ligate. Works fine ligating with
agarose. Typical ligation reaction is done in 40 ul - heat up the agaose bits
to 65 C first, mix with water from reaction mixture before adding buffers and 
ligase. Before transformation the ligation is again heated to 65 C, and then
put at 37 or 42 C for 5 min. Because of the bigger volume of ligation added
to the transformation, we add some buffer similar to that used for making your
competent cells (eg. for 40ul ligation, add 65 ul Ca buffer)

Marieke