Confusion with cloning strategy - (Jun/18/2005 )
Hey guys, I'm just sitting here and figure out a cloning strategy.
I've got a fragment of 9kb both on the 5' and on the 3' site a NheI restriction site.
Is it possible to clone this fragment into a 2.6kb vector with a unique NheI site within the Multiple cloning site. Is there anything I should be aware of?
theroretically this should be possible, but you should be aware that large plasmids (>7kb in my hands) are nasty to transform, so you may have some problems with your 11kb plamid. a good strategy is to try different cells and methods (chemical transformation vs electroporation).
i think it is possible to do that. just think another way around that your vector is about 9kb, while your insert is 2.5 kb. the only thing you have to be aware is the orientation after transformation. you can always cut it with a different enzyme which will cut both the insert and vector to check the orientation. i have done many single restriction site ligation. all of them work fine!!
You mentioned that transforming plasmid > 7kb is difficult. Why is that? What kind of cell do you use if you need to transform large plasmids?