Subcloning large fragments

S. S.
Fri Mar 24 23:04:39 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?
>
>Nick Ellison

I've cloned several large fragments into a Drosophila cloning vector
based on pUC18.  The largest plasmid I obtained was 21 kb, so 
cloning up to 18 kb is certainly possible.  I use DH5alfa.  The 
most serious problem is to transform the plasmid into bacteria.  I ALWAYS 
do these subcloning using a combination of two different restriction 
enzymes.  After ligation, whenever possible I digest the mixture
with an enzyme which shouldn't cut the desired construct.  I hope
it helps.  Cheers.  Slawek.



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