Ligating large fragments into pBluescript

pmiguel at bilbo.bio.purdue.edu pmiguel at bilbo.bio.purdue.edu
Fri May 9 15:27:07 EST 1997


In article <3.0.32.19970509135649.0070dd34 at imm2.imm.uth.tmc.edu>,
dhavilan at IMM2.IMM.UTH.TMC.EDU ("David L. Haviland, Ph.D.") wrote:

> 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

We routinely subclone lambda clone inserts into pBluescript (in the
NotI site).  12 kb is no problem.  Above 20 kb sometimes is difficult.
(The record for the lab is 32 kb.)  The Blue/White selection of pBluescript
works fine for this.  Phosphatasing the vector or using a limited amount
of vector will reduce the amount of blue colonies.  But even without this
it works okay usually.

Phillip SanMiguel



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