Help - Cloning Problems

Ted M Tedm at darkwing.uoregon.edu
Tue Jun 20 09:56:21 EST 1995


In article <3s6rf5$f9c at lastactionhero.rs.itd.umich.edu>, jpsmith at umich.edu
(James Smith) wrote:

 
> 
> If a plasmid has been treated with alkaline phosphatase, would it still 
> recircularize during ligation even if it does not take up the insert?
> 
> Should I be treating my DNA insert with alkaline phosphatase as well since
> both ends contain the XhoI restriction site?  If both DNA *and* plasmid
> are treated with alkaline phosphatase, will ligation still occur?

> 
> Thanks,
> Jim Smith
> jpsmith at umich.edu

Jim, in answer to your questions:
No, it shouldn't.
No, you shouldn't.
No, they won't.

The AP treatment is to avoid recircularization of the plasmid, which is
essential given the compatible cohesive ends a single Xho I cut will
generate. The phosphorylated insert will then be favored for ligation to
itself and the plasmid, although you will need to test for insert
orientation, of course. Its good to get rid of the AP after treating the
plasmid, a phenol extraction working well. Having both plasmid and insert
dephosphorylated will negate any ligation.
    The most probable problem is also one of the most common ones in
cloning; cutting near the ends of a fragment. If your insert had the Xho I
sites introduced via PCR or if the sites are too near the ends for any
reason, Xho I cuts very poorly. The NEB catalog contains valuable info on
this (and a lot more, its an invaluable ref.!). If your unsure of cutting,
a longer incubation may help.
Its also possible to follow end cutting with an end label of 32P. There
are even procedures (from Methods Enzym.) whereby you ligate the inserts
into long concatamers and then cut the (now) internal sites. This can be
handy as there is no need to engineer a more internal site (order new
primers etc). 
   It is also sometimes not neccessary to follow ligations via gels, do
you need high efficiency? or do you need ONE? Transforming will sometimes
give the desired clone, as the unligated DNA will not transform. Good
luck. 
                                                            Ted Michelini



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