Subcloning large fragments

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Thu Mar 23 09:13:15 EST 1995


In article <agpnwe-2303952007240001 at 161.66.230.5>, agpnwe at pnv.palm.cri.nz (Nick Ellison) writes:
>I need some advice on subcloning large fragments (15-20kb) from a lambda
>vector into a plasmid vector.  Is it possible to reliably subclone such
>fragments?  If so, what host/plasmid combinations are recommended?
>
I was not able to subclone the entire Not-insert from lambda FIXII clones,
but was successful many times, using Not and any other internal site.
Subcloning into double-cut plasmid worked well, but among the clones there
are often those with a reduced growth ( inserts were from human and
rat genomic DNA ), so you should look for smallish colonies and make sure
they are well purified from their neighbours. Some combinations of enzymes
resulted in cloning of the small arm into the plasmid, however, this clone
was stably  propagated, at least to the miniprep stage. I use XL-1 Blue
and several pUC-like vectors for that. My competent cells have the 
efficiency of more than 10E7 colonies per ug uncut vector, I think this is 
essential for cloning large fragments. Cheers,

*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
Universitaetstr.16
CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov



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