Subcloning into large plasmid - (Oct/19/2004 )

Hi,

what are optimal ligation conditions (especially temp. and time) for subcloning c. 4 kb insert into c. 16 kb plasmid (both cut with Not I & BstE II enzymes)?
Thank you for any suggestions or links.



Anetta

You can just follow whatever the ligase instruction says. Usually, you can do it at 4C overnight.

Hi! Just an extra advice. Do not purify your plasmid with gel extraction kit. Usually the colums don't work for plasmids > 10 kb.

Basically as for all ligation reactions a higher temperature leads to more efficient ligase activity, while a lower temperature allows a better annealing of the strands. Therefore most protocols start with a high temperature (usually around room temperature) with gradual reduction of temperature over night.
This can for instance be achieved in a standard thermo cycler. Alternatively one can fill a thermo flask with water, put your reaction tube in it (with an appropriate floater) and put the flask o/n in a fridge (4 °C). Due to the isolation the temperature will reduce slowly.