cloning of a big Plasmid - (Jul/25/2007 )
I need your help please.
I am trying a cloning experiment without succes!!!
Insert: 3657 bp
Vector: 5240 bp
Cut and Ligation was good
0,5 % TBE Agarose Gels
Transformation D5alpha E. coli´s + protocol from NEB
is the Plasmid (8897 bp) too big !!!!
no... i've dealed with 14kpb plasmid fine.
Just be sure that your cell are allow to well recover (at least 45' with Amp selection and 60' with kana selection.
Nope. Perfectly fine. Just watch out for the dephosphorylation step.
I have worked with 30kb plasmids.... 15kb inserts.
And my labmate, has worked with 90kb insert ligated into 70kb vector.
Just made some 12 kb plasmids. No problem with them.