Blunt-end cloning

Mikhail Alexeyev malexeye at JAGUAR1.USOUTHAL.EDU
Fri Aug 15 13:07:51 EST 1997


Lum wrote:
> 
> 
> 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.

In some of my clonings of short (up to 600 bp that is) PCR  products
Recombinant clones had a light blue color.  This happened only in
particular insert orientation.

On the other hand, I hope you did not phosphorylate your primers,
because if you did, then there is a good chance for insert
multimerization (high insert to vector ratio)which would mess up
everything.

M.A.



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