MBQ96AA at shef.ac.uk
Mon Aug 18 17:37:28 EST 1997
Dickens 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
> I'm very frustrated with this result and hope you can give me
> some suggestions. Thanks in advance...
I would suggest another idea for ligation. Using of antibiotic selection
might be good idea by using pBR322 digest it with EcoRV then you will get
blunt ends inside tetracyclin gene. Then you can ligate with your PCR
product and select these colonies which grow in Amp. plates but not in
Amp.+ Tet. plates
hopefully it will help
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