Cloning problem

Duncan Clark junk at hgmp.mrc.ac.uk
Mon Nov 11 08:11:08 EST 2002


Historians believe that in newspost <3DCF2611.C2E8B6B0 at uwo.ca> on Sun, 
10 Nov 2002, E Patterson <epatters at uwo.ca> penned the following literary 
masterpiece:
>If you're using just a a run of the mill plasmid, my first guess is
>you're REALLY not supposed to try to stick a 14kb insert into a 4kb
>plasmid.

It will go in but at lower efficiency than small inserts. ]

> From what I recall plasmids generally can only handle <7kb.

Nah, much more but ...

>Read the plasmids manual and it will probably say in the intro what the
>maximum size is.  You'll probably need a cosmid for something that's
>14kb.

It's quite often related to copy number of the plasmid, at this stage 
forgetting about toxic inserts.

Everybody uses pUC derivatives nowadays which means thousands of copies. 
Invariably, but not always, way too high for 14kb. pBR322 is better with 
say 25-50 copies depending upon which paper you read.

Below that you need say pSC101 derivatives with copy numbers of 1-2 or 
use the pLG series which have that copy number plus kan plus Blue/white 
from a paper in Gene maybe 15-20 years ago. The name Brian Spratt 
springs to mind, possibly then at University of Leicester.

If you can avoid blue white cloning it would help as you then don't need 
to have a strong promoter, lac, in front of your insert.

As to why 14kb won't go in? Lethal, too high a copy number, bad cloning, 
wrong E.coli host, wrong phase of the moon and so on.

Duncan
-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.



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