Ligating large fragments into pBluescript

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Fri May 9 13:55:01 EST 1997


At 14:41 5/9/97 +0000, Mark Leyland wrote:

>Has anybody got any experience of cloning quite large fragments of DNA
>into plasmid vectors?
>I'm trying to ligate fragments of 12kB and 18kb into pBluescript and
>just wondered whether I was simply wasting my time or if it was possible
>after all.
>Any comments, tips or advice welcome.

Mark:

Yes, I've done a bit.  I've ligated 12 and 14 kb fragments into pSP72.  For
both constructs, it took 2 ligation attempts for the 12kb and 3 for the
14kb insert.  Minipreps were useless as I had to screen by doing lifts and
probe that was contianed within my insert.

Ligation usually isn't the problem, transforming is.  With construct size,
efficiency falls off quickly.  With competent cells that gave mid-10^7
cfu/ug using bluescript only, gave about 10^3 cfu/ug with an intact cosmid
(40-ish kb) and about 10^5 cfu/ug with an intact bluebac vector at 10.5 kb.  

My rule of this is that if the total construct is going to exceed 10-12 kb,
then I'll opt for electroporation and use a bug such as TOP 10f' as it
accomodates large plasmids.

Just my $0.02 worth... hope it helps,
David




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