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Subcloning into large plasmid


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#1 Anetta

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Posted 19 October 2004 - 12:04 PM

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

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Posted 19 October 2004 - 05:38 PM

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

#3 raco

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Posted 26 October 2004 - 01:54 PM

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

#4 Charon

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Posted 27 October 2004 - 02:53 AM

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.




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