NotI and Star Activity

[Andrew Doherty]

Hi everyone out there,

Does anyone know if NotI shows appreciable 'star activity'? I've just
tried subcloning a cDNA from pcDNA1/amp to pcDNA3 with NotI/XbaI and
appear to have got pcDNA1/amp vector in my bugs!!!! The only way I can
see this happening is if NotI (GC!GGCCGC) has recognised an ApaI site
(GGGCC!C) and cut it to give NotI compatible ends. The pcDNA3 was
de-phosphorylated to reduce the chances of recircularisation of
incompletely digested vector, which would mean that my cDNA (NotI/XbaI
ends) could not clone into pcDNA3 (if NotI has cut at the ApaI site,
thus taking out the XbaI site), but pcDNA1/amp *could* recircularise if
my cDNA fragment was not completely separated from the vector. 

Is this feasible? Does anyone know how easily NotI recognises other
sites?

Thanks in advance


Andy D

-- 
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Dr Andrew Doherty		email -  a.doherty at bris.ac.uk
Dept. Anatomy			Tel (0117)9287421
School of Medical Sciences	Fax (0117)9287402
University of Bristol
University Walk
Bristol UK
BS8 1TD
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