PCR cloning

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Tue Mar 26 06:46:54 EST 2002


Dear Mika, 

You might use a high comeptency strain like XL10 from Stratagene 
(transformation efficiency is around 10^9 per ug of DNA) and several (=many) 
big agar plates. On one 10 cm dish, you may have around 10 to 50.000 clones at 
max.

But why do you need 10^8 clones? Can't you downscale your experiment or change 
the strategy?


> Hi,
> I try to get 100 million clones from a 900 Bp PCR insert. I use two RE:s
> at the ends of the PCR fragment (BglII and SalI) 

SalI is reported to be a bit nasty at the end of PCR products (NEB catalog) - did you add some extra bases at the primer? 

but I have obtained
> only 1000 clones with RT or 16 degrees ligations. Does anybody know how
> to scale-up the number of clones, or is 100 million clones just out of
> reach?

Regards, 
Wo
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Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan

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