Large plasmid cloning

b8703126 at b8703126 at
Sun Apr 4 02:50:21 EST 2004

I have a tough cloning. I try to clone a 3.8 kb insert (XhoI cut) into pshuttle vector (from Stratagene), which is also cutted by XhoI. And the size of this vector is about 8~9 kb. I have tried various ligation ratio and condition, but still got no colonies. The vector was linearized and dephosphorylated by CIP, and purified by gel-extraction. The insert was also purified by gel-extraction. And the ligation ratio i have tried was 2:1 and 3:1 (Insert: Vector). I do the ligation at 16 C, O/N. And the ligation volume is 10ul. Then i took 5ul of ligation product to transform DH5a. In the next day, i got no colonies on the kanamycin selective plates. Is there any thing wrong with my cloning procedures? Have anyone of u experienced this kind of situation? Please give me some suggestions, thanks a lot.

P.s. Could the concentration of kanamycin be a problem? Should i decrease the  concentration of kanamycin?

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