Large plasmid cloning
horner1978 at yahoo.co.uk
Tue May 4 14:00:36 EST 2004
I've been attempting some cloning since January with no luck. It turns out
that the Ligase I was using was inactive (Promega). You might want to check
yours by ligating Lambda DNA/Hind III fragments and then checking on a gel.
<b8703126 at sinamail.com> wrote in message
news:20040404075021.13400.qmail at ww02.hostica.com...
> 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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