Blunt-end cloning

Dom Spinella dspinella at chugaibio.com
Fri Aug 15 12:05:42 EST 1997


> Hi Netters,
> 
> I have several questions concerning the blunt-end cloning using
> the PCR-Script Blunt-end Cloning Kit from Stratagene.
> 
> I've performed five cloning reactions and the results
> are very unsatisfactory . My PCR products (300bp) are generated using
> the Pfu polymerase.
> It was then electrophoresed and eluted from gel using the Sephaglas
> Band-Prep Kit
> from Pharmacia. Ligation reaction is then followed exactly as described
> in the manual, with an insert:vector = 70:1, at 27C, using a new batch
> of Srf I enzyme just purchased fron Stratagene. Result: 30 ~ 40% of the
> colonies are white in color, with about 200 ~ 400 total colonies each
> plate. I've picked 30 white colonies (total 150 for five cloning
> reactions) for further analysis ( by restriction digestion of the
> extracted plasmid) but NONE of them contain the desire insert. ALL of
> them are FALSE positive.
> 
> Besides, some of the colonies are lightly blue in centre and
> white in peripheral. Are these colonies positive or negative clone..??
> In fact, some of the plates have none absolutely white colonies but
> these.
> 
> I'm very frustrated with this result and hope you can give me
> some suggestions. Thanks in advance...
> 
> Regards,
> Lum

Lum:

You have, of course, phosphorylated your PCR primers...???!!!  Just a 
thought.
-- D.G. Spinella



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