Subcloning large fragments
Fri Mar 24 23:04:39 EST 1995
In article <agpnwe-2303952007240001 at 18.104.22.168> 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'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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