speI xbaI sublconing

[Jose de las Heras]

"DK" <dk from no.email.thankstospam.net> wrote in message 
news:iFo6j.17$2R3.11 from newsfe02.lga...
> aawara from FEMA-trailer.org wrote:
>>In <vuy7ijqp4j1.fsf from shaggy.csail.mit.edu>,
>> Tom Knight <tk from csail.mit.edu> wrote:
>>
>>> "V Arunachalam" <vadivelarunachalam from yahoo.com> skrev i melding
>>> news:mailman.782.1196360538.23109.methods from net.bio.net...
>>>> I am facing problems in cohesive subcloning a 500 bp insert from the
>>>> double digest of SpeI and XbaI onto a vector of 13.5 Kb size at the 
>>>> same
>>>> sites. Please suggest me is it correct strategy as these two enzymes
>>>> generate similar ends though differ in recognition sites.
>>>
>>> Two options:
>>> 1) Ignore the problem and select one of the two orientations after the
>>>    fact.  About half should be in the orientation you want.
>>> 2) Add SpeI or XbaI to the ligation mix.  This will force the ligation
>>>    which recreates the SpeI or XbaI site to be cut, favoring the
>>>    ligation of the vector SpeI site with the insert XbaI site, and
>>>    vice-versa.
>>
>>SpeI and XbaI have compatible overhangs, and uni-molecular reactions are
>>always favored over bi-molecular reactions.
>>
>>Unless the original poster dephosphorylates the ends of his cut vector,
>>it will recircularize very efficiently, and none of his clones will 
>>contain
>>the insert.
>
> The situation is no different from blunt cloning. Easy to deal with by
> throwing a large excess of the insert. 20X or more.  With everything
> done properly, a reasonable percent of clones will contain an insert.
>
> DK

I'd agree with that and go for approach (1) above. Minipreps are easy and 
quick to do for screening, and usually the first handful give you at least 
one of the constructs you're looking for... I wouldn't worry too much, just 
mix the right components with a large excess of insert and screen for the 
correct one.

Jose
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